Devido à importância da agricultura para a economia do Brasil e para a dieta da população brasileira, um esforço contínuo tem sido feito para preservar a qualidade dos alimentos. Um dos mais importantes critérios de qualidade é a concentração correta dos pesticidas ou a não detecção destes nos alimentos. O objetivo deste trabalho é estudar o efeito matriz na analise de resíduos de pesticidas por cromatografia à gás em manga, a contaminação de mangas compradas na cidade de São Paulo por pesticidas e a otimização de um método multiresíduo neste tipo de matriz. O método empregado foi o utilizado pelo Governo Holandês adaptado de Lucke (1975), e constou da extração com acetona e partição em uma mistura diclorometano:hexano 1:1. O extrato foi concentrado e injetado em cromatógrafo à gás com detector de captura de elétrons e detector fotométrico de chama. Foram analisados 23 princípios ativos diferentes entre herbicidas, inseticidas, fungicidas e acaricidas; a metodologia empregada permitiu um limite de quantificação que variou de 0,01 mg/Kg a 0,30 mg/Kg. A recuperação de amostras fortificadas variou de 70 a 120%. Os resultados mostraram que a matriz apresenta uma grande influência na determinação dos princípios ativos Procloraz, Difenoconazol, Fosmete, Azoxistrobina e sulfato Endosulfam; o único princípio ativo encontrado nas amostras compradas no varejo foi o fungicida Procloraz com valores permitidos pela Legislação Brasileira. / Due to the importance of Agriculture for the economy of Brazil and for the diet of Brazilian population, a continuous effort is being made to preserve its high quality characteristics. One of the most important quality criteria is the right concentration or better not detectable pesticide residues. This work had the objective to study the matrix enhancement effect in gas chromatographic analysis of pesticide residue in mango fruit; the contamination of mango fruit by pesticide bought in Sao Paulo city and the optimization of the multiresidue method in this matrix. The employed method in the analysis was adapted from Lucke (1975), used by Netherlands Government; extraction with acetone and partition in a dichlorometane:hexane (1:1), and Gas Chromatographic determination with ECD and FPD. We studied about 23 active substances among herbicides, fungicides, insecticides and acaricides, the developed method allowed a quantification limit ranging from 0,01 mg/Kg to 0,30 mg/Kg. The recoveries of fortified samples ranged from 70 to 120%. The results showed that the matrix had a effect in the determination of the pesticides: prochoraz,difenoconazol, Fosmete, Azoxistrobina and Endosulfam Sulfate. It was found that the mango fruit were contaminated only by Procloraz; according to the Brazilian legislation, this pesticide can be present in mango.

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.

Das übergeordnete Ziel dieser Promotion war die Untersuchung und Kompensation des Matrixeffekts für die Analytik von Arzneimitteln in komplexen Probenmatrices mit LC-ESI-MS/MS-Technik. Zunächst konnte eine einfache analytische Methode für eine breite analytische Anwendbarkeit entwickelt werden. Es zeigte sich jedoch, dass die Matrixeffektkompensation zu einem Mehraufwand bei der Probenvorbereitung führt. Deshalb wurde anschließend der Mechanismus des Matrixeffektes auf die LC-ESI-MS/MS-Technik genauer untersucht. Die gewonnenen Erkenntnisse wurden anschließend eingesetzt, um eine einfache alternative Quantifizierungsmethode mittels der PCI eines internen Standards zu entwickeln. Im ersten Teilprojekt wurde eine LC-ESI-MS/MS-Methode für die Analytik von häufig verschriebenen Antibiotika in Abwasserproben der Stadt Dresden entwickelt. Da weder Vergleichsmatrix für Abwasser zur Verfügung stand, noch für alle Zielanalyte isotopenmarkierte Standards erhältlich sind, wurde der stark variierende Matrixeffekt der Abwasserproben mittels der Standardaddition kompensiert. Die Ergebnisse der Methodenentwicklung zeigen, dass eine genaue und flexible Methode entwickelt werden konnte, die Matrixkompensation jedoch zu einem erhöhten Zeit- und Materialaufwand führt. Es wurde deutlich, dass neben bisher genutzten Kompensationsmethoden für den Matrixeffekt, wie Standardaddition und interner isotopenmarkierter Standards, neue alternative Strategien getestet werden müssen. In dem zweiten Teilprojekt wurde daher der Matrixeffektmechanismus von Urin-, Plasma- und verschiedenen Abwasserproben bei der Messung von verschiedenen Arzneimitteln mittels LC-ESI-MS/MS analysiert. Die Ergebnisse der Untersuchungen mittels „post-column infusion“ konnten bisherige Erkenntnisse zu Matrixeffektmechanismen bestätigen und das Verständnis vertiefen. Matrixeffekte sind von der jeweiligen Zusammensetzung der Probenmatrix abhängig, aber auch substanzspezifisch. Dabei kommt es zwischen Analyt und Begleitsubstanzen zu einer Konkurrenz um freie Ladungsträger oder zu einer veränderten Anordnung/Verteilung innerhalb der ESI-Spray-Tröpfchen. Gleichzeitig zeigten die Ergebnisse, dass es auch andere Mechanismen, wie z. B. Ladungstransfers zwischen Analyt und Begleitsubstanzen, geben muss. Schließlich wurden die Ergebnisse des zweiten Teilprojekts in einer innovativen Methodenentwicklung zur Matrixkompensation und zur Quantifizierung von 16 Arzneimitteln in Urinproben verwendet. Der Matrixeffekt der Substanzen mit vergleichbarer Signalsuppression konnte über einen einzelnen nachsäuleninfundierten internen Standard kompensiert werden. Die Ergebnisse zeigen einen deutlichen Vorteil der entwickelten Methode gegenüber Matrix-Kalibrierung in Präzision und Richtigkeit oder dem Einsatz von isotopenmarkierten internen Standards in Aufwand der Methodenentwicklung und Verbrauch von Standardsubstanzen. Die Ergebnisse der vorliegenden Arbeit zeigen die Bedeutung, Komplexität und den Einfluss der Matrixeffekte in der Anwendung der LC-ESI-MS/MS-Technik. Einerseits sind geeignete Methoden für die Minimierung von Matrixeffekten wie Probenvorbereitung und Chromatographie nötig, andererseits müssen Ionisierungsmechanismen, insbesondere die Wechselwirkungen von Zielanalyten und Begleitsubstanzen, zukünftig Gegenstand weiterer Untersuchungen sein. Die Ergebnisse dieser Arbeit liefern wichtige Beiträge zur Verbesserung der Analytik von komplexen Proben mittels der LC-ESI-MS/MS-Technik. / The overall goal of this Ph.D. thesis was to investigate and compensate the matrix effect of the analysis of drugs in complex sample matrices with LC-ESI-MS/MS technique. First, a simple analytical method for a broad analytical applicability was developed for wastewater analysis. However, the matrix effect compensation embraced the main part effort in sample preparation. Therefore, the mechanism of the matrix effect on the LC-ESI-MS/MS technique was examined in more detail. The findings were used to develop an alternative quantification method using post-column infusion of an internal standard substance. In the first project, a LC-ESI-MS/MS method was developed to analyze commonly prescribed antibiotics in wastewater samples of Dresden. Since neither comparison matrix for wastewater nor all isotopically-labeled analogs for the target analytes were available, the strongly varying matrix effect of the wastewater samples was compensated by standard addition. The results show that the developed method is precise and flexible, but the matrix effect compensation leads to an increased expenditure of time and materials. Besides previously used matrix effect compensation methods, such as standard addition and internal isotopically-labeled standard, new alternative strategies need to be tested. Therefore, the matrix effect mechanism of various drugs and sample matrix combinations was examined in the second project using post-column infusion. The results confirmed previous findings on matrix effect mechanisms and deepened our understanding that matrix effects not only depend on the composition of the sample matrix but are also substance-specific. This results to a competition of free charge carriers between analyte and accompanying substances or to an alternated distribution within the ESI spray droplets. Furthermore, the results indicate that there are other mechanisms, such as charge transfer between analyte and concomitant substances. The results of the second project were used to invent a method for matrix effect compensation and quantification of 16 drugs in urine samples. The matrix effects of the substances with comparable signal suppression were compensated by a single post-column infused internal standard. The developed method has a significant advantage over the matrix calibration regarding precision and accuracy as well as the use of isotopically-labeled internal standards in effort of method development and consumption of standard substances. Finally, the results of this work show the importance, complexity and influence of the matrix effects in the application of the LC-ESI-MS/MS-technique. Suitable methods for minimizing matrix effects such as sample preparation and chromatography are needed and ionization mechanisms, in particular the interactions of target analytes with accompanying substances, should be investigated in future studies. The work of this Ph.D. project contributes to the improvement of the analysis of complex samples using the LC-ESI-MS/MS-technique.

Immunoassays basieren auf der Anwendung von Antikörpern, welche selektiv den zu messenden Analyten binden. Die Richtigkeit der erhaltenen Ergebnisse hängt maßgeblich von der Selektivität der Antikörper ab und kann durch Interferenzen gestört werden. In dieser Arbeit wurde eine Methode entwickelt, bei der die Probe mittels Hochleistungsflüssigkeitschromatographie (LC) in Fraktionen aufgetrennt wird und diese Fraktionen anschließend mittels Enzyme-linked Immunosorbent Assay (ELISA) vermessen werden. Dieses Verfahren wurde als LC-ELISA bezeichnet. Das erhaltene Profil aus im ELISA gemessener Analytkonzentration in Abhängigkeit von der Elutionszeit wurde als LC-ELISAgramm bezeichnet und bietet die Möglichkeit, Interferenzen zu erkennen, welche beim ELISA unentdeckt bleiben. Als Modellanalyten für die zu untersuchenden ELISAs dienten Sulfamethoxazol (SMX), Carbamazepin (CBZ) und Estron (E1). Dabei wurden verschiedene Umweltmatrices wie Oberflächenwasser und Abwässer mit dem jeweiligen ELISA vermessen. Es wurde ein Ansatz zur Unterscheidung von spezifischen und unspezifischen Interferenzen in Umweltproben aufgezeigt. Durch diesen Ansatz und Anwendung der sauren Hydrolyse der Probe war es möglich, einen bisher unbekannten SMX-Metaboliten zu detektieren und dessen wahrscheinliche Kreuzreaktivität mit 460 ± 150 % abzuschätzen. Es wurde zudem ein neuer Tracer in einer linearen 13-Stufen-Synthese entwickelt, wobei neuartig die Konjugation der Peroxidase an der N1-Position des SMX erfolgte. / Immunoassays are based on the use of antibodies that selectively bind the analyte. The trueness of the results obtained depends to a great extent on the selectivity of the antibodies and can be affected by interferences. In this study, a method was developed in which the sample is separated into fractions by using high-performance liquid chromatography (LC) and these fractions are measured using an enzyme-linked immunosorbent assay (ELISA). This method was referred to as LC-ELISA. The profile obtained from the measured analyte concentration by ELISA as a function of the elution time was referred to as LC-ELISAgram and offers the possibility to detect interferences which otherwise remain undetected during the ELISA. Sulfamethoxazole (SMX), carbamazepine (CBZ) and estrone (E1) were used as model analytes for the ELISA and LC-ELISA measurements. Various environmental matrices such as surface water and wastewater were examined for their interference in the respective ELISA. The good quantification properties of the validated LC-ELISA have been used to demonstrate an approach to distinguish between specific and non-specific interferences from environmental samples. By this approach and application of acidic hydrolysis of the sample, it was possible to detect a previously unknown metabolite of SMX and estimate its cross-reactivity to probably 460 ± 150%. Furthermore, a new tracer was developed in a linear 13-step synthesis, which resulted in the novel conjugation of the peroxidase at the N1-position of SMX. The new hapten was also used for the synthesis of a novel immunogen.