Schwefellost (SM) ist ein verbotener chemischer Kampfstoff. Nach Aufnahme über die Haut führt SM zu einer Reihe von Symptomen. Auf der molekularen Ebene beruht die Toxizität von SM auf der Reaktion mit DNA und Proteinen. Diese kovalenten Addukte eignen sich zum forensischen Nachweis und können über Flüssigkeitschromatographie gekoppelte Massenspektrometrie (LC-MS) detektiert werden. Ein Hauptziel war es in vitro zu untersuchen ob chemisches Scavenging von N-Acetylcystein (NAC) und Glutathion (GSH) Einfluss in der Behandlung von SM-Vergiftungen hat. Es konnte geklärt werden, dass NAC und GSH a) die Alkylierung von Cys34 in humanen Serum Albumin (HSA) durch SM nicht unterbinden kann und b) den Abbau von SM in gepufferter Lösung nicht beschleunigt. Somit ist Scavenging von SM durch NAC und GSH vernachlässigbar. Trotzdem konnte die Stabilität und die Zuverlässigkeit des Testsystems gezeigt werden. Das zweite Hauptziel war es neue peptidische Biomarker für den Nachweis von SM-Vergiftungen zu finden. Es wurde eine Methode entwickelt, um SM-alkylierte Aminosäurereste in HSA zu finden. Somit konnten 42 SM-alkylierte Peptide gefunden werden. Insgesamt wurden 27 Alkylierungsstellen identifiziert, von denen 24 noch nicht in der Literatur beschrieben wurden. Die vielversprechendste der neue Alkylierungsstellen war das Met329 in HSA. Durch Proteolyse mittels Pepsin wurde das LGM(-HETE)F Tetrapeptid freigeschnitten. Probenvorbereitung und LC-MS Bedingungen wurden optimiert. Allerdings gelang der Nachweis von LGM(-HETE)F in Patientenplasma nicht, was möglicherweise an einer geringeren Stabilität der SM-Alkylierung von Met329 lag. Trotzdem kann sich LGM(-HETE)F als Kurzzeitmarker eignen. Der beobachtete Transfer der HETE-Modifikation vom Met329 zu Glu und Cys Seitenketten stellt einen neuen Einblick in den molekularen Wirkmechanismus von SM dar. Basierend auf diesen Erkenntnissen konnte in Nachfolgestudien die Hemmung von Enzymen durch Alkylierung von Met durch SM gezeigt werden. / Sulfur mustard (SM) is a banned chemical warfare agent. After incorporation, SM may cause tremendous symptoms. On the molecular level, SM reacts with DNA and proteins. These covalent adducts may be useful for forensic or analytical purposes. After adducting SM, proteins can be proteolyzed forming peptides with a SM-modified amino acid residue. These peptide biomarkers can be detected using liquid chromatography-mass spectrometry (LC-MS). One major aim was to develop an in vitro assay to clarify the impact of chemical scavenging of N-acetylcysteine (NAC) and glutathione (GSH) in the treatment of SM. It was found that NAC and GSH had no relevant influence on a) the extent of alkylation of Cys34 in human serum albumin (HSA) and b) the degradation velocity of SM in buffered solution. Accordingly, it was concluded that chemical scavenging of SM by NAC or GSH is negligible. Nevertheless, the robustness and reliability of the developed in vitro assay was shown. The second major aim was to identify novel peptide biomarkers of SM poisoning. An untargeted method to identify SM-alkylated amino acid residues in HSA was developed. Application of this method revealed 42 SM-modified peptides alkylated at 27 different positions. 24 of these positions were not described in the literature so far. A highly interesting target, Met329 in HSA, was investigated in more detail. After pepsin mediated proteolysis, the covalently modified tetrapeptide LGM( HETE)F was generated. Sample preparation and LC-MS conditions were optimized. However, when applying this novel marker to real samples, it could not be detected likely due to its limited stability. Nevertheless, LGM( HETE)F still represents a suitable short term biomarker. The observed transfer of the HETE-moiety from Met329 to Glu and Cys side chains represents a novel insight into the molecular toxicology of SM. Based on these results follow-up studies proved the enzyme inhibitory effect of SM-alkylation of Met residues in keratin kinase.

<p>This thesis highlights the use and significance of surface enhanced Raman spectroscopy (SERS) for forensic applications. Two unique SERS substrates are developed for successful (1) forensic toxicological drug detection in human patient plasma and (2) trace explosive detection.  </p>

<p>Određivanje koncentracije etanola u telesnim tečnostima, pre svega u krvi, neophodan je uslov da bi se ustanovio uticaj alkoholemije na psihomotorne sposobnosti. Poznavanje stabilnosti lekova, droga i metabolita u biolo&scaron;kim uzorcima je od ključne važnosti kada se ukaže potreba za ponovljenom analizom i evaluacijom rezultata u sudskom postupku. Osnovni ciljevi ovog rada su da se uz pomoć HS-GC metode (hedspejs gasna hromatografija) ustanovi da li postoji statistički značajna promena koncentracije etanola u uzorcima krvi dobijenih od živih osoba i u biolo&scaron;kim uzorcima uzorcima sa autopsijskog materijala. Na osnovu rezultata potrebno je bilo utvrditi u kojem tipu uzorka uzetog sa le&scaron;nog materijala postoji najmanja promena koncentracije tokom perioda čuvanja uzorka. Istraživanje je bilo otvoreno, randomizirano i prospektivnog tipa. Biolo&scaron;ki uzorci krvi krvi živih osoba i le&scaron;nog materijala (krv, mokraća i staklasto telo) uzimani su metodom slučajnog izbora, u rasponu alkoholemije od 0,1 mg/ml do 5 mg/ml. Nakon inicijalne dvostruke analize, jedan biolo&scaron;ki uzorak čuvan je u trajanju od 180 dana, dok je drugi otvaran i analiziran nakon 60, 120 i 180 dana. Ukupan broj analiza alkoholemije u krvi živih osoba iznosio je 500. Ukupan broj analiza koncentracije etanola u krvi, mokraći i staklastom telu sa le&scaron;eva iznosio je 360. Etanol je u uzorcima krvi živih osoba, kao i u biolo&scaron;kim uzorcima sa autopsijskog materijala određivan metodom HS GC. Tokom čuvanja biolo&scaron;kih uzoraka u periodu od &scaron;est meseci ustanovljeno je da je do&scaron;lo do značajnog smanjenja koncentracije etanola u svim analiziranim uzorcima, nezavisno od njegovog porekla. Promena koncentracije etanola tokom čuvanja u zavisnosti je od tkivne vrste uzorka, inicijalne alkoholemije, dužine čuvanja, integriteta vijala i čepova, temperature, odnosa tečne i gasne faze, prisustva konzervansa i potencijalnog intermitentnog otvaranja radi analiza.</p> / <p>Determination of ethanol concentration in body fluids, especially blood, is a necessary objective to establish the influence of alcohol on psychomotor skills. Knowing the stability of medicines, drugs and metabolites in biological samples is of crucial importance when there is a need for repeated analysis and result evaluation in court. The main objectives of this work were to determine whether there was a statistically significant change in ethanol concentration in blood samples obtained from living subjects and from autopsy material, by using HS-GC method (headspace gas chromatography). Based on the results it was necessary to determine which type of sample collected from autopsy showed the lowest change in concentration during the storage period. The study was open, randomized and prospective. Biological samples of living person&#39;s blood and autopsy biological samples (blood, urine and the vitreous humor) were taken at random, in the level range between 0.1 mg/ml and 5 mg/ml. After an initial duplicate analysis, one biological sample was stored for a period of 180 days, while the other was opened and analyzed after 60, 120 and 180 days. Total number of analysis of living person&#39;s blood samples was 500. The total number of analysis of autopsy biological samples was 360. All concentrations were determined by HS-GC method. During the storage, results showed that there has been a significant decrease in the concentration of ethanol in all of the analyzed samples, regardless of its origin. The level of this change was dependent on the type of tissue sample, initial alcohol concentration, duration of storage, integrity of the vials and stoppers, temperature, ratio of liquid and gas phases, presence of preservatives and intermittent opening for analysis.</p>

Drug profiling, isto é, a caracterização de amostras de drogas apreendidas no sentido de estabelecer conexões entre apreensões realizadas em diferentes épocas e/ou locais a uma origem comum de produção clandestina, tem sido um objetivo dos órgãos governamentais responsáveis pela prevenção/repressão. Especificamente tratando-se de comprimidos de ecstasy, o conhecimento de suas propriedades físicas e químicas é de relevante importância para discriminar a apreensão de diferentes lotes. Nesse contexto, o presente trabalho propõe uma nova abordagem para estabelecer conexões entre apreensões de comprimidos de ecstasy, por meio da calorimetria exploratória diferencial (DSC), termogravimetria (TG) e difratometria de raios-X (DRX). Também foi realizada a caracterização física de todos os comprimidos (logotipo, coloração, massa, diâmetro e espessura), bem como a identificação/quantificação dos constituintes ativos por cromatografia em fase gasosa acoplada à espectrometria de massas (GC-MS) e o perfil de dissolução in vitro. Além disso, foi desenvolvido um método empregando a extração líquido-líquido para o isolamento da 3,4-metilenodioximetanfetamina (MDMA) dos comprimidos de ecstasy, que posteriormente foi cristalizada para cloridrato de MDMA (MDMA.HCl). Foram analisados dezessete diferentes lotes de comprimidos de ecstasy de diversos logotipos e colorações apreendidos no município de São Paulo, Brasil. Apenas um lote apresentou como única substância ativa a clorofenilpiperazina (CPP). Os outros continham apenas MDMA e o conteúdo de MDMA variou de 29 a 115-mg/comprimido. Os valores de massa dos comprimidos variaram de 143 a 341-mg, a espessura de 3,2 a 5,8-mm e o diâmetro de 7,0 a 9,5-mm. A comparação das curvas obtidas, tanto por calorimetria exploratória diferencial (DSC) como pelos difratogramas de raios-X (DRX), permitiu discriminar aqueles com perfis semelhantes, importante para identificar a origem de produção. O baixo grau de cristalinidade do MDMA.HCl de alguns comprimidos de ecstasy não impediu a caracterização por DSC e DRX. Esses resultados podem ser úteis para a aplicação no trabalho de inteligência forense. / Drug profiling or the characterization of seized drug samples to link seizures made at different times and/or locations to their common clandestine origin, has long been a goal of law enforcement agencies. Considering the trafficking of ecstasy tablets, the knowledge of chemical and physical properties is of utmost importance to discriminate between different seizures. In this context this study proposed a new approach to establish links among seizures of ecstasy tablets by using differential scanning calorimetry (DSC), thermogravimetry (TG) and X-ray diffraction (XRD). Besides this characterization, physical appearance (logotype, color, weight, diameter and thickness), identification/quantification of active constituents by gas chromatography/ mass spectrometry (GC/MS) and in vitro drug dissolution assays were performed too. A method employing liquid-liquid extraction was also developed for the isolation of 3,4-methylenedioxymethamphetamine (MDMA) from ecstasy tablets and afterwards MDMA was crystallized to MDMA hydrochloride (MDMA.HCl). Seventeen different lots of various logotypes and colors of confiscated ecstasy tablets from seizures in São Paulo city, Brazil, were analyzed. Chlorophenylpiperazine (CPP) was found only as an active ingredient in one batch. The others tablets contained only MDMA and the content of MDMA varied from 29 to 115-mg/tablet. The weight values of tablets varied from 143 to 341-mg, the thickness from 3,2 to 5,8-mm and the diameter from 7,0 to 9,5-mm. DSC/TG curves and X-ray difratograms of the ecstasy tablets allowed distinguishing those with similar profile, for both techniques, which is important to identify the source of production. The low degree of MDMA.HCl crystallinity of some ecstasy tablets didnt prevent DSC and XRD characterization. These results can be useful for forensic intelligence work application.

Os antidepressivos pertencem a uma importante classe de medicamentos investigados na toxicologia forense. Em casos de amostras provenientes de cadáveres, o intervalo entre o óbito e a obtenção da espécie biológica pode proporcionar a redistribuição postmortem destes fármacos. Com o objetivo de elucidar esse fenômeno, métodos analíticos foram desenvolvidos e aplicados utilizando sangue total (ST), humor vítreo (HV) e fígado. Para as amostras de ST e HV, o método de extração escolhido e validado foi a microextração em fase líquida (LPME) trifásica. Fibras ocas constituídas de polipropileno, com a extensão de 8 cm cada, foram tratadas com o solvente orgânico dodecano (fase orgânica), resultando em um membrana com permeabilidade seletiva. No lúmen destas fibras, adicionou-se ácido fórmico 0,1 mol/L (fase aceptora). Em frasco de fundo chato com 5 mL de capacidade, pipetou-se 3,5 mL de NaOH 0,1 mol/L (fase doadora) e 0,5 mL de ST ou HV. Ao término da extração, as amostras foram introduzidas no GC-MS, sem a necessidade de reações de derivatização. O estudo com ST contemplou os antidepressivos amitriptilina (AMI), nortriptilina (NTR), imipramina (IMI), desipramine (DES), clomipramina (CLO), desmetilclomipramina (DMC), fluoxetina (FLU) e norfluoxetina (NFL). Os limites de quantificação para estas substâncias ficaram inferiores aos níveis terapêuticos (20 ng/mL). As médias dos coeficientes de variação intradia e interdia foram, respectivamente, de 9,7 e 9,8%. As curvas de calibração apresentaram linearidade entre as concentrações de 20 até 1200 ng/mL. A validação do parâmetro integridade da diluição assegurou a mensuração de quantidades superiores ao limite apresentado na curva de calibração. O método foi aplicado em sete amostras reais postmortem e em apenas um caso foi observada uma diferença significativa (300%) entre os valores quantificados no ST periférico e central. Os antidepressivos tricíclicos AMI, NTR, IMI e DES foram avaliados no HV e o efeito matriz foi detectado para os dois últimos analitos. O método foi otimizado e validado utilizando solução salina adicionada de AMI e NTR. O limite de detecção igual a 5 ng/mL, foi obtido com a redução da voltagem da fonte de íons do espectrômetro de massa para 50 eV. Coeficientes de variação foram inferiores a 15%. Os procedimentos validados foram aplicados em seis amostras reais de HV. A relação encontrada entre os valores obtidos no ST periférico e HV foi de aproximadamente 0,1. A extração acelerada por solvente (ASE) e, posteriormente, a extração em fase sólida (SPE) foram as técnicas de separação dos analitos da matriz fígado. Ao término das citadas extrações, os antidepressivos foram analisados no GC-MS. Para esta matriz sólida, são necessários mais estudos, pois os valores encontrados nos ensaios analíticos estão em desacordo com as diretrizes utilizadas na validação dos métodos. / Antidepressants belong to an important class of drugs investigated in forensic toxicology. In cases of samples from corpses, the interval between death and obtaining the biological specimens can provide the postmortem redistribution of these drugs. Aiming to elucidate this phenomenon, analytical methods were developed and applied using whole blood (WB), vitreous humor (VH) and liver. For samples of WB and HV, the extraction method chosen and validated was the three-phase liquid phase microextraction (LPME). Hollow fibers consist of polypropylene, with a length of 8 cm each were treated with dodecane organic solvent (organic phase) resulting in a membrane with selective permeability. Into the lumen of these fibers was added formic acid 0.1 mol/ L (acceptor phase). In the vial containing 3.5 mL of NaOH 0.1 mol / L (donor phase) was spiked 0.5 ml of biological fluids (WB or VH). Subsequently, the samples were injected in GC-MS without derivatization reactions. The study of the ST included antidepressants amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI), desipramine (DES), clomipramine (CLO), desmethylclomipramine (DMC), fluoxetine (FLU) and norfluoxetine (NFL). The quantification limits for these substances were below the therapeutic levels (20 ng / ml). The mean coefficients of variation and separate intradays were respectively 9.7 and 9.8%. The calibration curves showed linearity between concentrations of 20 to 1200 ng / mL. The validation of the integrity of the dilution parameter assured measurement higher than the limit shown in the calibration curve quantities. The method was applied to seven real postmortem samples and in one case a significant difference (300%) between the measured values in the peripheral and central ST was observed. The tricyclic antidepressants AMI, NTR, IMI and DES were evaluated in VH and the matrix effect was detected in the last two analytes. The method was optimized and validated using saline spiked AMI and NTR. The limit of detection (5 ng/ml) was obtained by reducing the voltage of the ion source of the mass spectrometer 50 eV. Coefficients of variation were below 15%. The procedures were validated in six real samples of HV. The relationship found between the values obtained in the peripheral ST and HV was approximately 0.1. Accelerated solvent extraction (ASE) and subsequently the solid phase extraction (SPE) were the techniques of separation of analytes liver matrix. At the end of the cited extractions, antidepressants were analyzed in GC-MS. To this solid tissue, further studies are needed, because the values found in the analytical tests were not in accordance with the guidelines used in the validation of the methods.

Drug profiling, isto é, a caracterização de amostras de drogas apreendidas no sentido de estabelecer conexões entre apreensões realizadas em diferentes épocas e/ou locais a uma origem comum de produção clandestina, tem sido um objetivo dos órgãos governamentais responsáveis pela prevenção/repressão. Especificamente tratando-se de comprimidos de ecstasy, o conhecimento de suas propriedades físicas e químicas é de relevante importância para discriminar a apreensão de diferentes lotes. Nesse contexto, o presente trabalho propõe uma nova abordagem para estabelecer conexões entre apreensões de comprimidos de ecstasy, por meio da calorimetria exploratória diferencial (DSC), termogravimetria (TG) e difratometria de raios-X (DRX). Também foi realizada a caracterização física de todos os comprimidos (logotipo, coloração, massa, diâmetro e espessura), bem como a identificação/quantificação dos constituintes ativos por cromatografia em fase gasosa acoplada à espectrometria de massas (GC-MS) e o perfil de dissolução in vitro. Além disso, foi desenvolvido um método empregando a extração líquido-líquido para o isolamento da 3,4-metilenodioximetanfetamina (MDMA) dos comprimidos de ecstasy, que posteriormente foi cristalizada para cloridrato de MDMA (MDMA.HCl). Foram analisados dezessete diferentes lotes de comprimidos de ecstasy de diversos logotipos e colorações apreendidos no município de São Paulo, Brasil. Apenas um lote apresentou como única substância ativa a clorofenilpiperazina (CPP). Os outros continham apenas MDMA e o conteúdo de MDMA variou de 29 a 115-mg/comprimido. Os valores de massa dos comprimidos variaram de 143 a 341-mg, a espessura de 3,2 a 5,8-mm e o diâmetro de 7,0 a 9,5-mm. A comparação das curvas obtidas, tanto por calorimetria exploratória diferencial (DSC) como pelos difratogramas de raios-X (DRX), permitiu discriminar aqueles com perfis semelhantes, importante para identificar a origem de produção. O baixo grau de cristalinidade do MDMA.HCl de alguns comprimidos de ecstasy não impediu a caracterização por DSC e DRX. Esses resultados podem ser úteis para a aplicação no trabalho de inteligência forense. / Drug profiling or the characterization of seized drug samples to link seizures made at different times and/or locations to their common clandestine origin, has long been a goal of law enforcement agencies. Considering the trafficking of ecstasy tablets, the knowledge of chemical and physical properties is of utmost importance to discriminate between different seizures. In this context this study proposed a new approach to establish links among seizures of ecstasy tablets by using differential scanning calorimetry (DSC), thermogravimetry (TG) and X-ray diffraction (XRD). Besides this characterization, physical appearance (logotype, color, weight, diameter and thickness), identification/quantification of active constituents by gas chromatography/ mass spectrometry (GC/MS) and in vitro drug dissolution assays were performed too. A method employing liquid-liquid extraction was also developed for the isolation of 3,4-methylenedioxymethamphetamine (MDMA) from ecstasy tablets and afterwards MDMA was crystallized to MDMA hydrochloride (MDMA.HCl). Seventeen different lots of various logotypes and colors of confiscated ecstasy tablets from seizures in São Paulo city, Brazil, were analyzed. Chlorophenylpiperazine (CPP) was found only as an active ingredient in one batch. The others tablets contained only MDMA and the content of MDMA varied from 29 to 115-mg/tablet. The weight values of tablets varied from 143 to 341-mg, the thickness from 3,2 to 5,8-mm and the diameter from 7,0 to 9,5-mm. DSC/TG curves and X-ray difratograms of the ecstasy tablets allowed distinguishing those with similar profile, for both techniques, which is important to identify the source of production. The low degree of MDMA.HCl crystallinity of some ecstasy tablets didnt prevent DSC and XRD characterization. These results can be useful for forensic intelligence work application.

Os antidepressivos pertencem a uma importante classe de medicamentos investigados na toxicologia forense. Em casos de amostras provenientes de cadáveres, o intervalo entre o óbito e a obtenção da espécie biológica pode proporcionar a redistribuição postmortem destes fármacos. Com o objetivo de elucidar esse fenômeno, métodos analíticos foram desenvolvidos e aplicados utilizando sangue total (ST), humor vítreo (HV) e fígado. Para as amostras de ST e HV, o método de extração escolhido e validado foi a microextração em fase líquida (LPME) trifásica. Fibras ocas constituídas de polipropileno, com a extensão de 8 cm cada, foram tratadas com o solvente orgânico dodecano (fase orgânica), resultando em um membrana com permeabilidade seletiva. No lúmen destas fibras, adicionou-se ácido fórmico 0,1 mol/L (fase aceptora). Em frasco de fundo chato com 5 mL de capacidade, pipetou-se 3,5 mL de NaOH 0,1 mol/L (fase doadora) e 0,5 mL de ST ou HV. Ao término da extração, as amostras foram introduzidas no GC-MS, sem a necessidade de reações de derivatização. O estudo com ST contemplou os antidepressivos amitriptilina (AMI), nortriptilina (NTR), imipramina (IMI), desipramine (DES), clomipramina (CLO), desmetilclomipramina (DMC), fluoxetina (FLU) e norfluoxetina (NFL). Os limites de quantificação para estas substâncias ficaram inferiores aos níveis terapêuticos (20 ng/mL). As médias dos coeficientes de variação intradia e interdia foram, respectivamente, de 9,7 e 9,8%. As curvas de calibração apresentaram linearidade entre as concentrações de 20 até 1200 ng/mL. A validação do parâmetro integridade da diluição assegurou a mensuração de quantidades superiores ao limite apresentado na curva de calibração. O método foi aplicado em sete amostras reais postmortem e em apenas um caso foi observada uma diferença significativa (300%) entre os valores quantificados no ST periférico e central. Os antidepressivos tricíclicos AMI, NTR, IMI e DES foram avaliados no HV e o efeito matriz foi detectado para os dois últimos analitos. O método foi otimizado e validado utilizando solução salina adicionada de AMI e NTR. O limite de detecção igual a 5 ng/mL, foi obtido com a redução da voltagem da fonte de íons do espectrômetro de massa para 50 eV. Coeficientes de variação foram inferiores a 15%. Os procedimentos validados foram aplicados em seis amostras reais de HV. A relação encontrada entre os valores obtidos no ST periférico e HV foi de aproximadamente 0,1. A extração acelerada por solvente (ASE) e, posteriormente, a extração em fase sólida (SPE) foram as técnicas de separação dos analitos da matriz fígado. Ao término das citadas extrações, os antidepressivos foram analisados no GC-MS. Para esta matriz sólida, são necessários mais estudos, pois os valores encontrados nos ensaios analíticos estão em desacordo com as diretrizes utilizadas na validação dos métodos. / Antidepressants belong to an important class of drugs investigated in forensic toxicology. In cases of samples from corpses, the interval between death and obtaining the biological specimens can provide the postmortem redistribution of these drugs. Aiming to elucidate this phenomenon, analytical methods were developed and applied using whole blood (WB), vitreous humor (VH) and liver. For samples of WB and HV, the extraction method chosen and validated was the three-phase liquid phase microextraction (LPME). Hollow fibers consist of polypropylene, with a length of 8 cm each were treated with dodecane organic solvent (organic phase) resulting in a membrane with selective permeability. Into the lumen of these fibers was added formic acid 0.1 mol/ L (acceptor phase). In the vial containing 3.5 mL of NaOH 0.1 mol / L (donor phase) was spiked 0.5 ml of biological fluids (WB or VH). Subsequently, the samples were injected in GC-MS without derivatization reactions. The study of the ST included antidepressants amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI), desipramine (DES), clomipramine (CLO), desmethylclomipramine (DMC), fluoxetine (FLU) and norfluoxetine (NFL). The quantification limits for these substances were below the therapeutic levels (20 ng / ml). The mean coefficients of variation and separate intradays were respectively 9.7 and 9.8%. The calibration curves showed linearity between concentrations of 20 to 1200 ng / mL. The validation of the integrity of the dilution parameter assured measurement higher than the limit shown in the calibration curve quantities. The method was applied to seven real postmortem samples and in one case a significant difference (300%) between the measured values in the peripheral and central ST was observed. The tricyclic antidepressants AMI, NTR, IMI and DES were evaluated in VH and the matrix effect was detected in the last two analytes. The method was optimized and validated using saline spiked AMI and NTR. The limit of detection (5 ng/ml) was obtained by reducing the voltage of the ion source of the mass spectrometer 50 eV. Coefficients of variation were below 15%. The procedures were validated in six real samples of HV. The relationship found between the values obtained in the peripheral ST and HV was approximately 0.1. Accelerated solvent extraction (ASE) and subsequently the solid phase extraction (SPE) were the techniques of separation of analytes liver matrix. At the end of the cited extractions, antidepressants were analyzed in GC-MS. To this solid tissue, further studies are needed, because the values found in the analytical tests were not in accordance with the guidelines used in the validation of the methods.

<p>Ricin je prirodni protein, toksin koji spada među najpristupačnije i najsmrtonosnije otrove. Nalazi se u biljci Ricinus (Ricinus communis), sa najvećim sadržajem u semenu (1-5 %). Ricin se smatra potencijalnim bioterorističkim oružjem i prema riziku za ljudsko zdravlje svrstan je u B kategoriju biolo&scaron;kog oružja. U novije vreme kori&scaron;ćen je za konstruisanje imunotoksina protiv tumorskih ćelija u terapiji maligniteta. Dokumentovana su mnoga trovanja ricinom, kako zadesna, tako i samoubilačka i ubilačka. U tu svrhu koristilo se intaktno seme ricinusa ili ekstrahovani ricin. Osim ricina, u semenu ricinusa je prisutan toksični alkaloid ricinin u količini 0.3-0.8 %. Ricinus je jedini poznati prirodni izvor ricinina, koji se ko-ekstrahuje sa ricinom iz semena biljke. Ricinin se jednostavno detektuje u kliničkim uzoracima metodom tečne hromatografije i masene spektrometrije i, s obzirom na komplikovanu identifikaciju ricina u biolo&scaron;kim uzorcima, smatra se biomarkerom za intoksikaciju ricinusom, odnosno ricinom. Osnovni ciljevi ovog istraživanja su da se uz pomoć HS-GC metode i patohistolo&scaron;kom&nbsp; analizom dokaže prisustvo ricinina u krvi laboratorijskih pacova u odnosu na vremenski interval koji je protekao od oralne aplikacije suspenzije do vremena žtvovanja, da se odredi distribucija i koncentracija ricinina u organima laboratorijskih pacova u različitim vremenima žrtvovanja, kao i da se utvrdi da li postoji značajna razlika u razvoju patomorfolo&scaron;kih promena na organima laboratorijskih pacova u različitim vremenima žrtvovanja. Istraživanje je bilo otvoreno, randomizirano i prospektivnog tipa. Laboratorijski pacovi su u istom vremenu oralno tretirani suspenzijom koja je sadržaja subletalnu koncentraciju ricina. Nakon žrtvovanja u precizno definisanim vremenskim intervalima uzeti su uzorci krvi i unutra&scaron;njih organa radi daljih analiza. Odgovarajući uzorci su analizirani metodom HC-GS u cilju određivanja koncentracije i distribucije ricinina, kao pouzdanog markera trovanja ricinom, u krvi i unutra&scaron;njim organima. Takođe je izvr&scaron;ena patohistolo&scaron;ka analiza uzoraka tkiva unutra&scaron;njih organa u cilju utvrđivanja promena izazvanim delovanjem ricina u odnosu na vreme proteklo od aplikacije suspenzije. Dobijeni rezultati su obrađeni odgovarajućim statističkim metodama. Rezultati istraživanja omogućavaju standardizaciju postupaka odabira reprezentativnih uzoraka prilikom sumnje na trovanje ricinusom i metode dokazivanja akutnog trovanja. Na taj način može se pouzdano i efikasno dokazati trovanje ricinusom.</p> / <p>Ricin is a naturally occurring protein, a toxin which belongs to the category of the most accessible and the most lethal poisons. It is obtained from the castor oil plant ( Ricinus communis), whose seeds contain its highest content (1-5%). Ricin is also thought to be a potential weapon of bioterrorism and taking into account the risk for human health, it is classified as a biological weapon category B. Lately it has been used for the construction of the immunotoxins against tumor cells in the therapy of malignant diseases. Numerous poisonings using ricin have been documented, not only accidental poisoning, but also in case of suicides and homicides. In those cases, intact ricin seeds or extracted ricin were used. Apart from ricin, castor oil plants also contain a toxic alkaloid ricinine (0.3-0.8%). Castor oil plants are the only known natural source of ricinine, which is co-extracted with ricin from the seeds of this plant. Ricinine is simply detected in clinical samples by using the method of liquid chromatography and mass spectrometry. Taking into account a complicated identification of ricin in biological samples, it is considered to be a biomarker for the intoxication by castor oil plant, or ricin itself. The main aim of this research is to use the HS-GC method and pathohistological analysis in proving the existence of ricinine in the blood of experimental rats in relation to the time interval between the oral application of solution of castor seeds in water and the time of sacrificing, to determine the distribution and concentration of ricinine in the organs of experimental rats, as well as to establish whether there was a significant difference in the development of pathomorphological changes on the organs of experimental rats at various points of sacrificing. The research was open, randomised and prospective. Experimental rats were simultaneously orally tested by the solution which contained sublethal concentration of ricin. After sacrificing, blood samples were taken from inner organs in specifically defined intervals of time and used for further analysis. The appropriate samples were analysed by HC-GS method in order to determine the concentration and distribution of ricinine as a reliable marker of ricin poisoning in blood and inner organs. Also, pathohistological analysis of the samples of inner organ tissues was made with the purpose of establishing the changes caused by the effects of ricinine in relation to time which passed from the application of the solution. The obtained results were processed by appropriate statistical methods. The results of this research allow for the standardisation of the actions in selecting the representative samples in case there is a possibility of ricin poisoning and the method of proving the acute poisoning. Following these steps, ricin poisoning can be proved in a reliable and an efficient way.</p>