La douleur neuropathique centrale post accident vasculaire cérébral est une condition débilitante dont le traitement s’avère souvent délicat et infructueux. Le but de ce projet était de reproduire cette condition chez le rat en injectant par stéréotaxie une solution de collagènase produisant une hémorragie localisée dans le noyau ventropostérolatéral du thalamus. Des tests comportementaux évaluant la coordination motrice, la sensibilité mécanique, au chaud et au froid étaient réalisés régulièrement afin d’établir la présence de douleur neuropathique puis les effets de l’administration de kétamine, d’amitriptyline, de gabapentine, et de carbamazepine étaient évalués. L’induction d’une hémorragie intrathalamique conduit à l’apparition d’allodynie mécanique bilatérale persistante ainsi que d’allodynie au froid transitoire chez certains sujets et ce sans modification de la coordination motrice. L’administration de kétamine à forte dose renverse l’allodynie mécanique mais est associée à une altération de la motricité. L’administration de gabapentine renverse également cette allodynie mécanique sans effet notable sur la coordination motrice. Les autres médicaments n’ont pas démontré d’effet significatif. L’évaluation histopathologique des cerveaux montre une lésion bien localisée dans la zone d’intérêt. Ces résultats montrent que l’injection intrathalamique de collagénase peut être utilisée comme un modèle fiable de douleur neuropathique centrale. Si la kétamine semble capable de soulager ce type de douleur, elle est associée à des effets indésirables. En revanche, la gabapentine serait une molécule prometteuse pour le traitement de cette condition. Le rôle des récepteurs NMDA et des canaux calciques voltage dépendants, cibles respectives de la kétamine et de la gabapentine dans le maintien de cette douleur mérite d’être précisé. / Central post stroke pain is a debilitating condition for which treatment is often difficult and unsuccessful. The goal of this project was to reproduce an animal model of this condition by performing a stereotaxic injection of collagenase solution inducing a localized hemorrhage within the ventroposterolateral thalamic nuclei of rats. Behavioral tests to evaluate motor coordination, sentivity to mechanical, hot and cold stimuli were performed regurlarly to establish if neuropathic pain was present. Then, the effects of ketamine, amitriptyline, gabapentin and carbamazepine on such pain were evaluated. Induction of an intrathalamic hemorrhage produced bilateral persistent mechanical allodynia as well as transient cold allodynia in some animals without notable changes in motor coordination. High doses of ketamine did reverse mechanical allodynia but with significant impairment in motor coordination. Similarly gabapentin also reversed mechanical allodynia without undesirable effect on coordination. The others two drugs failed to show any significant effect. Histopathological evaluation of the brain revealed a well localized lesion in the area of interest. These results show that an intrathalamic collagenase injection produced a reliable model of central neuropathic pain. Ketamine was able to reverse mechanical allodynia but with significant side effects. Gabapentin appeared to be a promising drug for the treatment of this condition. The role of NMDA receptors and voltage dependant calcium channels, that target ketamine and gabapentin respectively in the maintenance of this pain, needs to be further defined.

In this thesis, rodent models of chronic pain were used to explore analgesic mechanisms that may potentially be engaged in spinal and peripheral compartments by systemically-administered amitriptyline, a tricyclic antidepressant. The first project (Chapter 2) identified the roles of spinal adenosine A1 and serotonin 5-HT7 receptors, as well as of peripheral adenosine A1 receptors, in the acute antinociceptive effects of amitriptyline in mice. The second project (Chapter 3) examined the potential utility of amitriptyline as a preventive analgesic against persistent post-surgical pain, and involved perioperative administration of amitriptyline after peripheral nerve injury in rats. Changes in post-injury behavioural outcomes, as well as spinal noradrenergic sprouting, were assessed. Overall, spinal serotonergic pathways linked to adenosine A1 receptors, as well as peripheral adenosine A1 receptors, appear to be important in antinociception by amitriptyline. Preventive analgesia by this drug does not appear to result from anatomical changes in spinal noradrenergic pathways.

Atlikta mokslinių literatūros šaltinių apžvalga. Tiriamųjų medžiagų identifikavimui ir kiekybinei analizei pritaikyta ir validuota efektyvioji skysčių chromatografija. Antidepresantų mišinys išskirtas iš kraujo plazmos kietos fazės ekstrakcijos metodu. KFE metodas optimizuotas keičiant eliuentą ir eliuento pH. Atliktas optimizacijos sąlygų palyginimas. Nustatyti 3 organiniai tirpikliai, kuriais efektyviausiai eliuuojamos sorbente sulaikytos tiriamosios medžiagos bei gauti statistiškai patikimi rezultatai (p>0,05) : 2 proc. etano rūgšties tirpalas 100 proc. metanolyje, 2 proc. etano rūgšties tirpalas 80 proc. metanolyje ir 2 proc. metano rūgšties tirpalas 100 proc. metanolyje. / In this thesis was performed research of scientific literature. Applied and validated high performance liquid chromatography for identification and quantification of target compounds. Antidepressant mixture extracted from blood plasma samples using LLE and SPE methods. SPE method was optimized by changing the elutor and its pH. Three organic solvents were determined, which best elute target compounds from sorbent and show statistical confidence (p>0,05): 2 proc. acetic acid in 100 proc. methanol solution, 2 proc. acetic acid in 80 proc. methanol solution and 2 proc. formic acid in 100 proc. methanol solution.

No description available.

Atualmente, a quantidade de pacientes que são diagnosticados com alguma forma de depressão, entre elas, o transtorno depressivo maior, aumenta consideravelmente, quer seja em razão de diagnósticos mais precisos ou pela própria epidemiologia da doença. Acresça-se o fato de que muitos pacientes, apesar da quantidade de tipos de antidepressivos atualmente disponíveis para a terapêutica, são refratários ao tratamento prescrito, em razão dos efeitos adversos apresentados ou ainda em razão de simplesmente não se observar melhora com a prescrição. Em razão disso, novos tratamentos farmacológicos são disponibilizados. Para auxiliar na máxima eficácia em sua utilização, esse trabalho propôs o desenvolvimento de metodologia analítica para a determinação simultânea de antidepressivos tricíclicos e não tricíclicos, a saber: moclobemida, venlafaxina, citalopram, agomelatina, duloxetina, amitriptilina e sertralina em plasma humano por cromatografia líquida de alta eficiência (HPLC), para posteriormente ser aplicada na monitorização de pacientes depressivos. O método consistiu na extração líquido-líquido com recuperação entre 73% a 86%, exceto para a moclobemida (55%). A separação foi obtida usando uma coluna em fase reversa LiChrospher® 60 RP-select B em LichroCART 250mm x 4mm, 5 ?m de diâmetro interno, Merck sob condições isocráticas com detecção em UV em 230 nm, com fase móvel composta por 35% de uma mistura de acetonitrila/metanol 55/5 v/v e 65% de tampão acetato 0,25M pH 4,4. As curvas padrão foram lineares em uma faixa de trabalho de 2,5-1000 ng/mL para moclobemida, 5-2000 ng/mL para venlafaxina, citalopram, agomelatina, duloxetina e amitriptilina, 10-2000 ng/mL para sertralina. A precisão intra e interensaios foi efetuada em 3 concentrações (50, 200 e 500 ng/mL). Os coeficientes de variação para a precisão intraensaio foram menores que 8,8% para todos os compostos e os coeficientes de variação para a precisão interensaios foram menores que 8,6%. Os limites de quantificação foram de 2,5 ng/mL para a moclobemida, 5 ng/mL para venlafaxina, citalopram, duloxetina, agomelatina, amitriptilina e 10 ng/mL para sertralina com a etidocaína como padrão interno. Não se observou qualquer interferência das drogas normalmente associadas com antidepressivos. O método desenvolvido foi aplicado na monitorização de 79 pacientes em tratamento prolongado com amitriptilina, sertralina e venlafaxina. Paralelamente, foram avaliadas as concentrações plasmáticas de pacientes voluntários sadios submetidos a tratamento em dose única com agomelatina. A monitorização terapêutica se faz necessária para monitorar adesão ao tratamento já que a depressão está entre as principais causas mundiais de morbidade por incapacitação social e estimativa de prevalência crescente até 2030; caracterizando-se como um dos maiores e mais onerosos problemas de saúde pública. / Currently, the number of patients who are diagnosed with some form of depression, among them, major depressive disorder, increases considerably, either because of more accurate diagnosis or by the epidemiology of the disease. One should add the fact that many patients, despite the amount of types of antidepressants currently available for therapy are refractory to the treatment prescribed, because of the adverse effects appear, or their toxic effects, or by simply not observed improvement then the prescription. Therefore, new pharmacological treatments are available. To assist in maximum effectiveness in its use, this paper presents a methodology on HPLC for simultaneous determination of seven antidepressants, tricyclic and non-tricyclic, moclobemide, venlafaxine, citalopram, agomelatine, duloxetine, amitriptyline and sertraline in human plasma, later to be applied in monitoring depressed patients. The simple and accurate method of sample preparation consists of the liquid-liquid extraction with recovery between 73% to 86% (except for moclobemide, 55%). Separation was achieved using a reverse phase column Lichrospher® 60 RP-select B LiChroCART 4mm x 250mm, 5?m internal diameter, Merck, under isocratic conditions, with UV detection at 230nm, with a mobile phase consisting of a mixture of 35% acetonitrile:methanol 55/5 (v/v) and 65% 0.25M acetate buffer, pH 4.4. The standard curves were linear in the working range of 2,5-1000 ng/mL for moclobemide, 5-2000 ng/mL to venlafaxine, citalopram, agomelatine, duloxetine and amitriptyline and 10-2000ng/mL to sertraline. The intra and interassay precisions were performed at three concentrations (50, 200 and 500 ng/mL). The coefficients of variation for intra-assay precision were less than 8.6% for all compounds and the coefficients of variation for interassay precision were lower than 8.5%. The limits of quantification were 2.5 ng/mL for moclobemide, 5 ng/mL for venlafaxine, citalopram, duloxetine, agomelatine, amitriptyline and 10 ng/mL for sertraline. No interference of drugs normally associated with antidepressants was observed. The developed method was applied to the monitoring of 79 patients receiving prolonged treatment with amitriptyline, sertraline and venlafaxine. And, the plasma concentrations of healthy volunteer patients undergoing single dose agomelatine were evaluated. Therapeutic drug monitoring, is to ensure maximum clinical efficacy coupled with minimal adverse effects in patients undergoing long-term therapy is needed and to monitor adherence to treatment since depression is among the major causes of morbidity and social disability increasing prevalence estimate of 2030; characterized as one of the largest and most costly public health problems

Irritable bowel syndrome (IBS) är en av de vanligaste diagnostiserade GI-tillstånden idag och är en störning av gastrointestinalkanalen (GI-kanalen). Det finns inget botemedel mot IBS på grund av att patogenesen är oklar och därför är fokusen symtomlindring vid behandling.  De vanligaste symtom som förekommer är buksmärta, obehag i buk, uppblåsthet, utspänd buk och förändring av avföringens konsistens och frekvens. Patofysiologin är inte fullt klarlagd men en rubbning i tarm-hjärn-axeln kan leda till förändring i GI-rörelser. Neurotransmittorerna noradrenalin och serotonin (5-HT) är troligen viktiga faktorer för patofysiologin kopplad till tarm-hjärn-axeln. Syftet var att undersöka effektiviteten och säkerheten för de antidepressiva läkemedlen vid behandling av IBS. De antidepressiva som undersöktes i arbetet var tianeptin, amitriptylin, escitalopram, venlafaxin, vortioxetin och mirtazapin. De fem artiklarna som användes i detta arbete upptäcktes med hjälp av databaserna Pubmed och Onesearch. Sökord som användes var ”IBS” AND ”antidepressants” och ”IBS” AND ”SSRI”.  Alla antidepressiva visade en signifikant förbättring för livskvalitén hos deltagarna i studierna. Tianeptin, amitriptylin, escitalopram, venlafaxin och mirtazapin undersökte förändring av buksmärta och alla hade en signifikant förbättring förutom escitalopram. Escitalopram jämfördes mot rektal ballongutvidgning vid behandling hos IBS med förstoppning (IBS-C). Ballongutvidgningen visade bättre resultat än escitalopram och denna jämförelse gör det svårt att dra någon slutsats om escitalopram i arbetet. Amitriptylin, tianeptin och mirtazapin undersökte förändring av avföringskonsistensesn och frekvensen och visade en signifikant förbättring hos IBS med diarré (IBS-D). Venlafaxin visade signifikant förbättring för både lös och hård avföringsfrekvens och studerade ingen specifik IBS-grupp. Vortioxetin undersökte alla IBS-grupper och endast förändring av livskvalitén, depression och ångest vilket också gör det svårt att dra slutsats om effekt hos IBS. Det var inga av de depressiva medlen som gav allvarliga biverkningar, dock kan vissa biverkningar tolkas som mer obehagliga än andra. För vissa antidepressiva var det deltagare som avslutade studien på grund av biverkningar. Amitriptylin, tianeptin, venlafaxin vortioxetin och mirtazapin visade alla god effekt och säkerhet. Om effektiviteten ock säkerheten jämförs bland dessa har tianeptin det bästa resultatet. / Irritable bowel syndrome (IBS) is one of the most diagnosed conditions in the gastrointestinal (GI) tract today and is a disorder of the GI tract. There is no cure for IBS, which is probably due to the fact that the pathogenesis is unclear, and therefore the focus has been a symptom-relieving treatment. The most common symptoms that occur are abdominal pain, abdominal discomfort, bloating, distension of the abdomen, and change in the consistency and frequency of the stool. The pathophysiology is not fully understood, but a disorder of the gut-brain axis can lead to a change in GI movements. The neurotransmitters norepinephrine and serotonin (5-HT) are probably important factors for pathophysiology and linked to the gut-brain axis.  The aim was to investigate the effectiveness and safety of antidepressants used in IBS treatment. The antidepressants examined in this literature were tianeptine, amitriptyline, escitalopram, venlafaxine, vortioxetine, and mirtazapine. The five articles on which current litter tour work is based were obtained using the PubMed and OneSearch databases. Keywords used were "IBS" AND "antidepressants" and "IBS" AND "SSRI."  All the antidepressants examined showed a significant improvement in the participants' quality of life in the studies. The studies also examined changes in abdominal pain using tianeptine, amitriptyline, escitalopram, venlafaxine, and mirtazapine. All participants showed a significant improvement and reduced abdominal pain except when ingesting escitalopram. Intake of escitalopram was compared against rectal balloon enlargement as a treatment for IBS with constipation (IBS-C). The balloon expansion showed better results than escitalopram, and this comparison makes it difficult to draw any conclusion about escitalopram and its actual effect in different types of IBS. Amitriptyline, tianeptine, and mirtazapine investigated stool consistency and stool frequency changes and showed a significant improvement in these symptoms in IBS with diarrhea (IBS-D). Venlafaxine showed significant improvement for both loose and hard stool frequency; however, no specific IBS group was studied in this study. When ingesting vortioxetine, all different IBS groups and changes in quality of life, depression, and anxiety were examined, making it difficult to conclude about the effect of the drug in IBS. None of the antidepressant medicines produced severe side effects. However, some side effects can be interpreted as more unpleasant than others, and hence there was some loss during some studies.  Amitriptyline, tianeptine, venlafaxine vortioxetine, and mirtazapine showed good efficacy and safety. If the effectiveness and safety were to be compared between these drugs, tianeptine would be the first choice in treating IBS.

Background: Antidepressants are widely used in the treatment of chronic pain. Applied doses are lower than those needed to unfold an antidepressive effect. While efficacy of antidepressants for chronic pain has been reported in large randomized-controlled trials (RCT), there is inconsistent data on adverse effects and tolerability. We aimed at synthesizing data from RCT to explore adverse effect profiles and tolerability of antidepressants for treatment of chronic pain. Methods: Systematic literature research and meta-analyses were performed regarding side effects and safety of different antidepressants in the treatment of chronic pain according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The National Center for Biotechnology Information library and MEDLINE were searched. Randomized placebo-controlled trials were included in quantitative data synthesis. results: Out of 1,975 screened articles, 33 papers published between 1995 and 2015 were included in our review and 23 studies were included in the meta-analyses. A higher risk for adverse effects compared to placebo was observed in all antidepressants included in our analyses, except nortriptyline. The most prevalent adverse effects were dry mouth, dizziness, nausea, headache, and constipation. Amitriptyline, mirtazapine, desipramine, venlafaxine, fluoxetine, and nortriptyline showed the highest placebo effect-adjusted risk of adverse effects. Risk for withdrawal due to adverse effects was highest in desipramine (risk ratio: 4.09, 95%-confidence interval [1.31; 12.82]) followed by milnacipran, venlafaxine, and duloxetine. The most common adverse effects under treatment with antidepressants were dry mouth, dizziness, nausea, headache, and constipation followed by palpitations, sweating, and drowsiness. However, overall tolerability was high. Each antidepressant showed distinct risk profiles of adverse effects. conclusion: Our synthesized data analysis confirmed overall tolerability of low-dose antidepressants for the treatment of chronic pain and revealed drug specific risk profiles. This encompassing characterization of adverse effect profiles might be useful in defining multimodal treatment regimens for chronic pain which also consider patients’ comorbidities and co-medication.

Background: Antidepressants are widely used in the treatment of chronic pain. Applied doses are lower than those needed to unfold an antidepressive effect. While efficacy of antidepressants for chronic pain has been reported in large randomized-controlled trials (RCT), there is inconsistent data on adverse effects and tolerability. We aimed at synthesizing data from RCT to explore adverse effect profiles and tolerability of antidepressants for treatment of chronic pain. Methods: Systematic literature research and meta-analyses were performed regarding side effects and safety of different antidepressants in the treatment of chronic pain according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The National Center for Biotechnology Information library and MEDLINE were searched. Randomized placebo-controlled trials were included in quantitative data synthesis. results: Out of 1,975 screened articles, 33 papers published between 1995 and 2015 were included in our review and 23 studies were included in the meta-analyses. A higher risk for adverse effects compared to placebo was observed in all antidepressants included in our analyses, except nortriptyline. The most prevalent adverse effects were dry mouth, dizziness, nausea, headache, and constipation. Amitriptyline, mirtazapine, desipramine, venlafaxine, fluoxetine, and nortriptyline showed the highest placebo effect-adjusted risk of adverse effects. Risk for withdrawal due to adverse effects was highest in desipramine (risk ratio: 4.09, 95%-confidence interval [1.31; 12.82]) followed by milnacipran, venlafaxine, and duloxetine. The most common adverse effects under treatment with antidepressants were dry mouth, dizziness, nausea, headache, and constipation followed by palpitations, sweating, and drowsiness. However, overall tolerability was high. Each antidepressant showed distinct risk profiles of adverse effects. conclusion: Our synthesized data analysis confirmed overall tolerability of low-dose antidepressants for the treatment of chronic pain and revealed drug specific risk profiles. This encompassing characterization of adverse effect profiles might be useful in defining multimodal treatment regimens for chronic pain which also consider patients’ comorbidities and co-medication.

[pt] A derivatização fotoquímica foi utilizada para induzir ou amplificar a fluorescência de três substâncias utilizadas como princípios ativos em medicamentos: tetrabenazina (TBZ), amitriptilina (AMT) e vareniclina (VRN). Com o aumento de fluorescência após o tratamento com radiação UV, métodos espectrofluorimétricos foram desenvolvidos para determinar a concentração de TBZ, a AMT e a VRN em medicamentos e em matrizes biológicas. A avaliação da abordagem por cromatografia líquida de alta eficiência (HPLC) com detecção por fluorescência tambme foi avaliada para a AMT e VRN. Para a determinação de TBZ, a irradiação por 60 min em meio de NaOH (0,45 mol L-1) permitiu a sua quantificação espectrofluorimétrica com um limite de detecção (LD) de 1,4 x 10-8 mol L-1 e faixa linear até 1,0 x 10-5 mol L-1. O método espectrofluorimétrico foi aplicado para determinar TBZ no medicamento com recuperações entre 88 e 95%. A TBZ também foi determinada em saliva fortificada com o analito em concentração próxima ao do limite de quantificação (LQ) com recuperações maiores que 87%. Estudos usando espectrometria de massas com desorção de plasma estimulada por uma fonte de 252Cf foram realizados para identificar a estrutura do fotoderivado, indicando a fotólise da TBZ via reação de Norrish tipo I e II. Para a determinação de AMT, duas abordagens de derivatização fotoquímica permitiram determinar a substância por espectrofluorimetria e por HPLC. Para o método espectrofluorimétrico, a derivatização foi realizada expondo ao UV por 60 min uma solução de AMT, preparada em HCl 0,027 mol L-1. A faixa de resposta linear se estendeu até 1,0 x 10-5 mol L-1 de AMT com um LD de 4,8 x 10-8 mol L-1. Na abordagem cromatográfica, o processo de derivatização da AMT foi ajustado utilizando 30 min de UV em tampão fosfato pH 3. O método permitiu um LD de 6,9 x 10-9 mol L-1 com faixa linear até 2,5 x 10-6 mol L-1 de AMT. Os métodos desenvolvidos foram aplicados na determinação de AMT em medicamento e em material de referência farmacopéico com recuperações entre 94 e 105% para o método fluorimétrico e entre 99 e 111% para a abordagem cromatográfica. A aplicação na determinação da AMT em plasma humano fortificado na concentração de 1,0 x 10-6 mol L-1 forneceram resultados aceitáveis. Os fotoderivados fluorescentes foram separados e identificados por GC-MS, sendo proposto um mecanismo de fotólise da AMT. Para a determinação de VRN, a derivatização fotoquímica em meio básico permitiu a determinação da substância por espectrofluorimetria, sendo também avaliada uma abordagem por HPLC. A derivatização da VRN, realizada expondo o analito ao UV por 23 min em solução de NaOH 0,64 mol L-1, permitiu um LD de 1,5 x 10-8 mol L-1 e uma faixa linear até 1,0 x 10-6 mol L-1. As condições de fotoderivatização também foram adaptadas para a determinação cromatográfica do analito, irradiando-o por 10 min em meio de tampão borato (0,01 mol L-1 pH 10). A aplicação na determinação da VRN no medicamento com recuperações entre 93 e 102% foi alcançada. Para todos os métodos desenvolvidos foi realizada uma análise metrológica simplificada contemplando as fontes de incerteza mais relevantes para o processo de medição dos analitos: o preparo de soluções; a repetibilidade; a precisão intermediária e a curva analítica. As incertezas de medição para cada um dos métodos propostos foram estimadas em três concentrações distintas, no início, no meio e no fim da faixa linear. Os resultados obtidos foram entre 5,8 e 36% de incerteza do valor nominal das concentrações próximas ao LQ e entre 3,1 e 16% para as concentrações dentro na faixa linear dos métodos. Em todos os métodos desenvolvidos foram utilizados procedimentos de derivatização seguros, energeticamente eficientes e com redução de uso de solventes e substâncias tóxicas, em concordância com os princípios da química verde. / [en] Photochemical derivatization was employed to induce or amplify the fluorescence from three substances with pharmacological activities: tetrabenazine (TBZ); amitriptyline (AMT) and varenicline (VRN). The substantial fluorescence enhancement after UV irradiation allowed the development of methods based on spectrofluorimetry for TBZ, AMT and VRN. The approach was also evaluated in high performance liquid chromatography (HPLC) with fluorimetric detection for the determination of the concentration AMT and VRN in drugs or in biological matrices. For the determination of TBZ, 60 min of UV irradiation of the analyte prepared in NaOH (0.45 mol L-1) allowed its indirect quantification using spectrofluorimetry, with a limit of detection (LOD) of 1.4 x 10-8 mol L-1 and a linear response up to 1.0 x 10-5 mol L-1. Pre-concentration using solid phase extraction improved the LOD in one order of magnitude. The spectrofluorimetric method was applied for the determination of TBZ in a commercial drug, with recoveries between 88 and 95%. TBZ was also determined in analyte fortified saliva at a concentration near the limit of quantitation (LOQ) with recoveries greater than 87%. Studies using plasma desorption mass spectrometry with a 252Cf source, indicated that the photoderivatives of TBZ were obtained through a Norrish Type I and II TBZ photolysis. For the determination of AMT, two photochemical derivatization approaches allowed the determination of the substance by spectrofluorimetry. The approach using HPLC coupled to fluorescence detection was also tested. For the spectrofluorimetric method, the derivatization was achieved after 60 min of UV exposure of a solution of AMT prepared in HCl 0.027 mol L-1. The linear response of the method was up to 1.0 x 10-5 mol L-1 of AMT, with a LOD of 4.8 x 10-8 mol L-1. For the chromatographic method, the derivatization process was adjusted (30 min of UV irradiation in phosphate buffer, pH 3) in order to adapt the reaction for HPLC. The method allowed an LOD of 6.0 x10-9 mol L-1 with a linear range up to 2.5 x 10-6 mol L-1 of AMT. The determination of AMT in drugs and in a pharmacopeial reference material was performed, with recoveries between 94 and 105% for the fluorimetric method and between 99 and 111% using HPLC. The analysis of human plasma was also made using AMT (1.0 x 10-6 mol L-1) fortified samples. The fluorescent photoderivatives were separated and identified by GC-MS, which allowed the proposal of a photolysis mechanism of AMT. For the determination of VRN, the photochemical derivatization in basic medium allowed the determination of the substance by spectrofluorimetry. The derivatization of VRN, performed by exposing the analyte to the UV during 23 minutes in NaOH 0.64 mol L-1, allowed a LOD of 1.5 x10-8 mol L-1 and a linear range extending up to 1.0 x10-6 mol L-1. The derivatization condition were also adapted for the determination of the analyte using HPLC with fluorimetric detection, by irradiating VRN for 10 min in borate buffer (0.01 mol L-1 pH 10). Both methods were applied for the determination of VRN in a commercial drug, with recoveries between 93 and 102%. For all the developed methods, a simplified metrological evaluation was performed, considering the most relevant sources of uncertainty during the measurement process of the analytes studied in this thesis: solution preparation, intra and inter-day precision and the analytical curve. The uncertainty sources for each of the proposed methods were estimated in three different concentrations within the linear range. Measurement uncertainty estimations were between 5.8 and 36% of the nominal concentration in the concentrations near the LOQ and between 3.1 and 16% in the concentrations within the linear range of the methods. Safer, energetically efficient procedures with solvent reduction and without the use of toxic reagents were proposed, in agreement to green chemistry principles.