Methods for polyamine derivatization with a panel of extrinsic fluorophores followed by HPLC with fluorescence and UV absorption detection have been developed. Four fluorophores were examined using polyamines and aminoglycosides. o-Phthalaldehyde (OPA) and fluorescamine are selective fluorophores that only react with primary amines; 9- fluorenylmethyl chloroformate (FMOC Cl) and dansyl chloride react with both primary and secondary amines. Reaction and HPLC conditions were optimized with each of the above fluorophores using a series of model mono- and diamines and then applied to natural and semi-synthetic polyamines. The amines that have been investigated are natural di- and polyamines: putrescine, cadaverine, spermidine, spermine, thermospermine, aminoglycosides: kanamycin, paramomycin, neomycin, and synthetic polyamine conjugates e.g. N⁴,N⁹-dioleoylspermine, N¹-cholesteryl spermine carbamate. The resultant derivatives were confirmed by off-line high resolution electrospray ionization mass spectrometry (HR ESI MS). The results show that the synthesis of polyamine derivatives in quantitative yield depends on the time of reaction, the temperature and the ratio of fluorophore reagent. Linearity of derivatization was calculated and regression coefficients ranged from 0.968 to 0.999 with good reproducibility. HR ESI MS analysis of the reaction products demonstrated complete derivatization of both primary and secondary amino groups with dansyl and FMOC fluorescence derivatives and of primary amine groups for OPA and fluorescamine derivatives. Under the ionization conditions used the dansyl derivatives showed, in addition to monovalent ions [M+H]⁺, divalent cations [M+2H]²⁺ because this chromophore contains a basic amine that can be easily protonated. FMOC derivatives gave prominent [M+Na]⁺ ions. The OPA derivatization reaction is rapid, but the products have poor stability. The derivatization with fluorescamine gave multiple products with glucosamine due to the presence of a chiral centre in the fluorophore. The relative quantum yields of the polyaminefluorophore derivatives were examined to determine the effect of intramolecular fluorescence quenching. Dansylation is the fluorescent derivatization method of choice.

Nos últimos anos, a pesquisa ambiental tem se defrontado com a questão dos chamados disruptores endócrinos (EDCs). A estes compostos, tais como: produtos farmacêuticos, hormônios naturais e sintéticos, pesticidas, substâncias tensoativas, polímeros de baixa massa molecular e diversos outros contaminantes orgânicos presentes em efluentes municipais e industriais, atribui-se à capacidade de alterar o funcionamento do sistema endócrino. Estrogênios e progestogênios, naturais ou sintéticos, são excretados pela urina de mamíferos, e uma pequena porção nas fezes, e via efluentes de estações de tratamento de esgoto (ETE) entram em vias aquáticas, podendo causar alterações em organismos aquáticos, tais como feminização ou hermafroditismo. Neste contexto, no presente trabalho foi descrito uma metodologia analítica para a extração em fase sólida empregando cartucho C18, dos hormônios naturais, estrona (E1) e 17?-estradiol (E2), e dos hormônios sintéticos, levonorgestrel e 17?-etinilestradiol (EE2) (presentes em anticoncepcionais orais), a partir de uma matriz de esgoto sintético. Foram utilizados dois sistemas cromatográficos neste estudo, ambos de mesmo modelo (SLC-10A, Shimadzu), os quais consistiram em um injetor manual (seringa), com um volume de injeção ajustado para 20µL, e duas bombas modelo LC-10ADVP (Shimadzu). Foram utilizados dois tipos de detectores, um sistema DAD modelo SPD-M10AVP (Shimadzu) e um espectrofluorímetro modelo RF-551 versão 2.4 (Shimadzu). A separação foi feita em coluna C18 (250 X 4,6 mm, 5 µm) com um fluxo de 1 mL min-1. A condição ideal para separação foi o modo isocrático: 48/52 ACN:H2O. O comprimento de onda selecionado para quantificação no sistema de detecção DAD foi: 240nm para o levonorgestrel e 280nm para E1, E2 e EE2. No detector espectrofluorímetro, os comprimentos de onda selecionados para a excitação e emissão dos analitos E1, E2 e EE2 foram: 280nm e 306nm, respectivamente. Para se efetuar o estudo de recuperação, uma matriz simulando esgotos sanitários foi utilizada com o intuito de se ter uma amostra controle (testemunha) livre dos analitos de interesse, devido à dificuldade em se obter uma amostra de esgoto “real" livre destes hormônios. As amostras de esgoto sintético foram fortificadas em três níveis de concentração. Tomou-se 5 replicatas de 100,0mL de amostra testemunha (esgoto sintético) para cada nível de fortificação, estas amostras foram dopadas com os hormônios estudados. Após adaptações de metodologias descritas na literatura, a extração foi feita segundo o procedimento: condicionamento do cartucho (500mg/6mL) com 7mL de acetonitrila, 5mL de metanol e 5mL de água em uma razão de fluxo de 3mL min-1; percolagem de 250,0mL de amostras de esgoto bruto e efluentes tratados pela ETE-Araraquara com fluxo de 1mL min-1; secagem do cartucho por 1 hora a vácuo; eluição dos analitos com 6mL de acetonitrila com fluxo de 1mL min-1; secagem do extrato eluído em corrente de nitrogênio; reconstituição da amostra em 0,5mL de metanol. As amostras de esgoto sintético foram dopadas com os padrões estudados, para análise realizada no sistema DAD, a 0,250?g L-1 para levonorgestrel e 2,50?g L-1 para E1, E2 e EE2 (nível 1); 0,375 ?g L-1 para levonorgestrel e 3,75 ?g L-1 para E1, E2 e EE2 (nível 2); 0,500?g L-1 para levonorgestrel e 5,00?g L-1 para E1, E2 e EE2 (nível 3). Para análise realizada no sistema fluorescente, as amostras de esgoto sintético foram dopadas com os padrões estudados a 0,750?g L-1 para E1; 0,150?g L-1 para E2 e 0,250?g L-1 para EE2 (nível 1); 1,00 ?g L-1 para E1; 0,150?g L-1 para E2 e 0,200?g L-1 para EE2 (nível 2); 1,25?g L-1 para E1; 0,250?g L-1 para E2 e 0,300 ?g L-1 para EE2 (nível 3). Valores de recuperação entre 83-123% com coeficientes de variação menores do que 13,5% foram obtidos para todos os hormônios analisados pelos dois sistemas de detecção (DAD e Fluorescente). Esses dados demonstram a eficiência do método quanto à exatidão e precisão para os níveis de fortificação estudados. O método proposto foi utilizado para avaliar a presenças dos hormônios em afluentes (esgoto bruto) e efluentes da ETE-Araraquara. As amostras coletadas na ETE foram analisadas em triplicata. Foi identificado e quantificado o hormônio natural E2 (31ng L-1) em amostras obtidas antes do tratamento de esgoto. Não foram detectadas concentrações dos analitos em amostras obtidas após o tratamento. / In recent years environmental research has been faced with the issue of the endocrine disrupting chemicals (EDCs). Such compounds, such as: pharmaceutical products, natural and synthetic hormones, pesticides, tensive active substances, low mass molar polymers and many other organic contaminants that appear in municipal and industrial effluents, have the capacity of altering the manner in which the endocrine system works. Natural or synthetic estrogens and progestogens are excreted through the urine of mammals, and a small portion through faeces, and via effluents from sewage treatment plants (STP) flow into aquatic ducts, with the possibility of causing alterations in the aquatic organisms, such as feminization or hermaphroditism. Within this context, the present work describes an analytic methodology for solid phase extraction (SPE) using C18 cartridge of the natural hormones, estrone (E1) and 17?-estradiol (E2), and the synthetic hormones levonorgestrel and 17?-ethinylestradiol (EE2) (found in oral contraceptives) from a synthetic waste matrix. Two chromatographic systems were used in this study, both from the same model (SLC-10A, Shimadzu), a DAD system model SPD-M10AVP (Shimadzu) and a spectrofluorimeter model RF-551 type 2.4 (Shimadzu). Separation was performed in column C18 (250 X 4,6 mm, 5 ?m) with a flux of 1 mL min-1. The ideal separation condition was the isocratic mode: 48/52 ACN:H2O. To carry out the recuperation study, a matrix simulating sewers was used with the objective of having a control sample (witness) free of the samples under scrutiny of the difficulty in obtaining a “real" sewage sample free of these hormones. The samples of synthetic sewage were boosted in three levels of concentration. Recuperation values between 83-123% with variation coefficients lower than 13,5% were obtained for all studied hormones by both systems of detections (DAD and Fluorescent). These data demonstrate the efficiency of the method concerning the accuracy and precision for the fortification levels that were studied. The proposed method was used to assess the presence of hormones in inffluents (raw sewage) and effluents of Araraquara-STP. The natural hormone E2 (31ng L-1) was identified and quantified in samples obtained prior to sewage treatment. No concentrations of analytes in the samples were obtained after the treatment.

Nos últimos anos, a pesquisa ambiental tem se defrontado com a questão dos chamados disruptores endócrinos (EDCs). A estes compostos, tais como: produtos farmacêuticos, hormônios naturais e sintéticos, pesticidas, substâncias tensoativas, polímeros de baixa massa molecular e diversos outros contaminantes orgânicos presentes em efluentes municipais e industriais, atribui-se à capacidade de alterar o funcionamento do sistema endócrino. Estrogênios e progestogênios, naturais ou sintéticos, são excretados pela urina de mamíferos, e uma pequena porção nas fezes, e via efluentes de estações de tratamento de esgoto (ETE) entram em vias aquáticas, podendo causar alterações em organismos aquáticos, tais como feminização ou hermafroditismo. Neste contexto, no presente trabalho foi descrito uma metodologia analítica para a extração em fase sólida empregando cartucho C18, dos hormônios naturais, estrona (E1) e 17?-estradiol (E2), e dos hormônios sintéticos, levonorgestrel e 17?-etinilestradiol (EE2) (presentes em anticoncepcionais orais), a partir de uma matriz de esgoto sintético. Foram utilizados dois sistemas cromatográficos neste estudo, ambos de mesmo modelo (SLC-10A, Shimadzu), os quais consistiram em um injetor manual (seringa), com um volume de injeção ajustado para 20µL, e duas bombas modelo LC-10ADVP (Shimadzu). Foram utilizados dois tipos de detectores, um sistema DAD modelo SPD-M10AVP (Shimadzu) e um espectrofluorímetro modelo RF-551 versão 2.4 (Shimadzu). A separação foi feita em coluna C18 (250 X 4,6 mm, 5 µm) com um fluxo de 1 mL min-1. A condição ideal para separação foi o modo isocrático: 48/52 ACN:H2O. O comprimento de onda selecionado para quantificação no sistema de detecção DAD foi: 240nm para o levonorgestrel e 280nm para E1, E2 e EE2. No detector espectrofluorímetro, os comprimentos de onda selecionados para a excitação e emissão dos analitos E1, E2 e EE2 foram: 280nm e 306nm, respectivamente. Para se efetuar o estudo de recuperação, uma matriz simulando esgotos sanitários foi utilizada com o intuito de se ter uma amostra controle (testemunha) livre dos analitos de interesse, devido à dificuldade em se obter uma amostra de esgoto “real” livre destes hormônios. As amostras de esgoto sintético foram fortificadas em três níveis de concentração. Tomou-se 5 replicatas de 100,0mL de amostra testemunha (esgoto sintético) para cada nível de fortificação, estas amostras foram dopadas com os hormônios estudados. Após adaptações de metodologias descritas na literatura, a extração foi feita segundo o procedimento: condicionamento do cartucho (500mg/6mL) com 7mL de acetonitrila, 5mL de metanol e 5mL de água em uma razão de fluxo de 3mL min-1; percolagem de 250,0mL de amostras de esgoto bruto e efluentes tratados pela ETE-Araraquara com fluxo de 1mL min-1; secagem do cartucho por 1 hora a vácuo; eluição dos analitos com 6mL de acetonitrila com fluxo de 1mL min-1; secagem do extrato eluído em corrente de nitrogênio; reconstituição da amostra em 0,5mL de metanol. As amostras de esgoto sintético foram dopadas com os padrões estudados, para análise realizada no sistema DAD, a 0,250?g L-1 para levonorgestrel e 2,50?g L-1 para E1, E2 e EE2 (nível 1); 0,375 ?g L-1 para levonorgestrel e 3,75 ?g L-1 para E1, E2 e EE2 (nível 2); 0,500?g L-1 para levonorgestrel e 5,00?g L-1 para E1, E2 e EE2 (nível 3). Para análise realizada no sistema fluorescente, as amostras de esgoto sintético foram dopadas com os padrões estudados a 0,750?g L-1 para E1; 0,150?g L-1 para E2 e 0,250?g L-1 para EE2 (nível 1); 1,00 ?g L-1 para E1; 0,150?g L-1 para E2 e 0,200?g L-1 para EE2 (nível 2); 1,25?g L-1 para E1; 0,250?g L-1 para E2 e 0,300 ?g L-1 para EE2 (nível 3). Valores de recuperação entre 83-123% com coeficientes de variação menores do que 13,5% foram obtidos para todos os hormônios analisados pelos dois sistemas de detecção (DAD e Fluorescente). Esses dados demonstram a eficiência do método quanto à exatidão e precisão para os níveis de fortificação estudados. O método proposto foi utilizado para avaliar a presenças dos hormônios em afluentes (esgoto bruto) e efluentes da ETE-Araraquara. As amostras coletadas na ETE foram analisadas em triplicata. Foi identificado e quantificado o hormônio natural E2 (31ng L-1) em amostras obtidas antes do tratamento de esgoto. Não foram detectadas concentrações dos analitos em amostras obtidas após o tratamento. / In recent years environmental research has been faced with the issue of the endocrine disrupting chemicals (EDCs). Such compounds, such as: pharmaceutical products, natural and synthetic hormones, pesticides, tensive active substances, low mass molar polymers and many other organic contaminants that appear in municipal and industrial effluents, have the capacity of altering the manner in which the endocrine system works. Natural or synthetic estrogens and progestogens are excreted through the urine of mammals, and a small portion through faeces, and via effluents from sewage treatment plants (STP) flow into aquatic ducts, with the possibility of causing alterations in the aquatic organisms, such as feminization or hermaphroditism. Within this context, the present work describes an analytic methodology for solid phase extraction (SPE) using C18 cartridge of the natural hormones, estrone (E1) and 17?-estradiol (E2), and the synthetic hormones levonorgestrel and 17?-ethinylestradiol (EE2) (found in oral contraceptives) from a synthetic waste matrix. Two chromatographic systems were used in this study, both from the same model (SLC-10A, Shimadzu), a DAD system model SPD-M10AVP (Shimadzu) and a spectrofluorimeter model RF-551 type 2.4 (Shimadzu). Separation was performed in column C18 (250 X 4,6 mm, 5 ?m) with a flux of 1 mL min-1. The ideal separation condition was the isocratic mode: 48/52 ACN:H2O. To carry out the recuperation study, a matrix simulating sewers was used with the objective of having a control sample (witness) free of the samples under scrutiny of the difficulty in obtaining a “real” sewage sample free of these hormones. The samples of synthetic sewage were boosted in three levels of concentration. Recuperation values between 83-123% with variation coefficients lower than 13,5% were obtained for all studied hormones by both systems of detections (DAD and Fluorescent). These data demonstrate the efficiency of the method concerning the accuracy and precision for the fortification levels that were studied. The proposed method was used to assess the presence of hormones in inffluents (raw sewage) and effluents of Araraquara-STP. The natural hormone E2 (31ng L-1) was identified and quantified in samples obtained prior to sewage treatment. No concentrations of analytes in the samples were obtained after the treatment.

In vivo intracranial microdialysis is a valuable technique yielding novel and useful insight into normal or pathological neurochemical processes in the brain by means of sampling of interstitial fluid of cells in a living animal. It's most important advantage is that it can continuously monitor time-related changes in the concentration of neurotransmitters and their metabolites, other neuromodulators, energy substrates, as well as exogenous drugs in the extracellular fluid of specific brain areas of interest. While the development and standardization of the intracranial microdialysis technique in our laboratory was the main aim of the current study, a pilot application study was also performed during which the effect of several locally administered pharmacological agents on brain glutamate levels in a genetic rat model of depression was investigated. Abnormal neuronal glutamate levels have been implicated in various psychiatric conditions including major depressive disorder. The Flinders Sensitive Line (FSL) is a genetic line of Sprague-Dawley rat that displays various behavioral and neurochemical traits akin to that observed in depression. The Flinders Resistant Line (FRL) rat is used as the normal control. The prefrontal cortex is an important brain area involved in the neuropathology of depression. Prefrontal cortical glutamate levels in a small number of FSL and FRL rats were therefore compared at baseline and following local administration of potassium chloride (100 mM), the latter in order to study changes in evoked glutamate release. Ketamine hydrochloride (9 mM) and fluoxetine (30 μM) respectively were also administered via reverse dialysis. Prior to initiating the microdialysis studies, an HPLC-fluorescence method was developed to analyze the levels of glutamate in the microdialysate. As part of the development and standardization of the microdialysis technique, a number of validation studies were performed. This included refining the stereotaxic surgery procedure, determining the most appropriate anesthesia protocol, and standardizing the microdialysis procedure with regard to perfusion fluid, flow rate, sample volume, duration of dialysis, and anatomical verification of probe location. The HPLC-fluorescence method for the analysis of glutamate was also developed and validated. This technique proved to be sensitive and specific for the determination of glutamate with a linearity of 0.991 in the concentration range of standards tested (0.1 – 10 μM) and an intra-assay repeatability (precision value) yielding relative standard deviations of less than 10.5%, Mean elution time was between 24 and 26 minutes for glutamate in the microdialysis sample and the limit of detection and quantification was both 0.1 μM. Results from the application study indicated that baseline values of glutamate in the prefrontal cortex did not differ between FRL and FSL rats during the 1 hour period of dialysis. However, potassium chloride-evoked glutamate release was greater in FSL vs. FRL rats, although this difference was not statistically significant. Local perfusion by reverse dialysis of ketamine hydrochloride produced statistically significant increases in glutamate concentrations at certain time points in FSL rats. Although glutamate levels were also increased in FRL rats in response to ketamine, it was not statistically different compared to baseline levels. Fluoxetine perfusion did not affect glutamate release in either of the two rat groups. In conclusion, we have successfully developed and established an intracranial in vivo microdialysis procedure in our laboratory, as well as standardized and validated a sensitive method to analyze glutamate in microdialysate samples. These techniques were then applied in a small number of FSL vs. FRL rats in order to confirm their application in a typical research scenario. Although the data were too limited to make any valid conclusions about glutamate concentrations in an animal model of depression or the effect of drugs on the release thereof, these novel techniques and analyses will be valuable in future studies. / Thesis (MSc (Pharmacology))--North-West University, Potchefstroom Campus, 2013.

In vivo intracranial microdialysis is a valuable technique yielding novel and useful insight into normal or pathological neurochemical processes in the brain by means of sampling of interstitial fluid of cells in a living animal. It's most important advantage is that it can continuously monitor time-related changes in the concentration of neurotransmitters and their metabolites, other neuromodulators, energy substrates, as well as exogenous drugs in the extracellular fluid of specific brain areas of interest. While the development and standardization of the intracranial microdialysis technique in our laboratory was the main aim of the current study, a pilot application study was also performed during which the effect of several locally administered pharmacological agents on brain glutamate levels in a genetic rat model of depression was investigated. Abnormal neuronal glutamate levels have been implicated in various psychiatric conditions including major depressive disorder. The Flinders Sensitive Line (FSL) is a genetic line of Sprague-Dawley rat that displays various behavioral and neurochemical traits akin to that observed in depression. The Flinders Resistant Line (FRL) rat is used as the normal control. The prefrontal cortex is an important brain area involved in the neuropathology of depression. Prefrontal cortical glutamate levels in a small number of FSL and FRL rats were therefore compared at baseline and following local administration of potassium chloride (100 mM), the latter in order to study changes in evoked glutamate release. Ketamine hydrochloride (9 mM) and fluoxetine (30 μM) respectively were also administered via reverse dialysis. Prior to initiating the microdialysis studies, an HPLC-fluorescence method was developed to analyze the levels of glutamate in the microdialysate. As part of the development and standardization of the microdialysis technique, a number of validation studies were performed. This included refining the stereotaxic surgery procedure, determining the most appropriate anesthesia protocol, and standardizing the microdialysis procedure with regard to perfusion fluid, flow rate, sample volume, duration of dialysis, and anatomical verification of probe location. The HPLC-fluorescence method for the analysis of glutamate was also developed and validated. This technique proved to be sensitive and specific for the determination of glutamate with a linearity of 0.991 in the concentration range of standards tested (0.1 – 10 μM) and an intra-assay repeatability (precision value) yielding relative standard deviations of less than 10.5%, Mean elution time was between 24 and 26 minutes for glutamate in the microdialysis sample and the limit of detection and quantification was both 0.1 μM. Results from the application study indicated that baseline values of glutamate in the prefrontal cortex did not differ between FRL and FSL rats during the 1 hour period of dialysis. However, potassium chloride-evoked glutamate release was greater in FSL vs. FRL rats, although this difference was not statistically significant. Local perfusion by reverse dialysis of ketamine hydrochloride produced statistically significant increases in glutamate concentrations at certain time points in FSL rats. Although glutamate levels were also increased in FRL rats in response to ketamine, it was not statistically different compared to baseline levels. Fluoxetine perfusion did not affect glutamate release in either of the two rat groups. In conclusion, we have successfully developed and established an intracranial in vivo microdialysis procedure in our laboratory, as well as standardized and validated a sensitive method to analyze glutamate in microdialysate samples. These techniques were then applied in a small number of FSL vs. FRL rats in order to confirm their application in a typical research scenario. Although the data were too limited to make any valid conclusions about glutamate concentrations in an animal model of depression or the effect of drugs on the release thereof, these novel techniques and analyses will be valuable in future studies. / Thesis (MSc (Pharmacology))--North-West University, Potchefstroom Campus, 2013.