O uso de drogas por mulheres em idade reprodutiva é considerado um grave problema de saúde pública mundial. O mecônio é a primeira excreção do recém-nascido e tem sido utilizado como uma matriz alternativa em análises toxicológicas. A extração em fase sólida (SPE) é um método amplamente utilizado para a purificação e concentração dos analitos em amostras biológicas no campo da análise forense. A maioria dos produtos de SPE convencionais requerem volumes relativamente grandes de solventes levando a um custo acrescido por amostra e um aumento no tempo de processamento da amostra. As ponteiras de extração com fase sólida modificada (DPX) foram utilizadas como uma alternativa aos cartuchos SPE tradicionais. A técnica combina eficiência e rapidez no procedimento de extração, com redução significativa no consumo de solvente e na quantidade de amostra. O objetivo desse estudo foi desenvolver e validar um método para a determinação de nicotina, cotinina, cocaína, benzoilecgonina, cocaetileno e éster metil anidroecgonina em amostras de mecônio usando DPX e cromatografia em fase gasosa acoplada à espectrometria de massa (GC/MS). Os resultados da validação indicaram uma extração eficiente, exata e precisa, com recuperação entre 50-98%, exatidão entre 92-106%, precisão intra-ensaio 4-12% e precisão inter-ensaio 6 a 12%. As curvas de calibração foram lineares com valores de R2 superiores a 0,99; os limites de detecção (LOD) variaram entre 2,5-15 ng/g e os limites de quantificação (LOQ) entre 10-20 ng/g. O método DPX-GC/MS mostrou ser eficaz para análise traços de drogas presentes em amostras de mecônio. Após desenvolvimento e validação, o método foi aplicado em 50 amostras de mecônio coletadas no berçário da Maternidade do Complexo Aeroporto (MATER) na cidade de Ribeirão Preto - São Paulo, Brasil. / Drug abuse by pregnant women is considered a serious public health problem worldwide. Meconium is the first excretion in newborns and has been used as an alternative matrix to evaluate in utero drug exposure. Solid phase extraction (SPE) is widely employed to prepare and cleanup samples in the field of forensic analysis. Most SPE products require large volumes of solvent, which culminates in longer sample processing times; increased cost per sample and higher limits of detection. Disposable Pipette Extraction tips (DPX) have been used as an alternative to traditional SPE cartridges. They combine efficient and rapid extraction with reduced solvent consumption. The purpose of this study was to develop and validate a method to determine nicotine, cotinine, cocaine, benzoylecgonine, cocaethylene and methyl ester anhydroecgonine in meconium using DPX and GC/MS. Validation results indicated extraction efficiency, ranged between 50-98%, accuracy 92-106%, intra-assay precision 4-12% and inter-assay precision 6 to 12%. Linear calibration curves resulted in R2 values greater than 0.99; limits of detection ranged from 2.5 - 15 ng/g and the limit of quantitation from 10 - 20 ng/g. The DPX-GC/MS method provided to selectively analyze trace concentrations of drugs in meconium samples. Finally, the developed and validated method was applied to 50 meconium samples collected at the nursery of Maternidade do Complexo Aeroporto (MATER) in the city of Ribeirão Preto - São Paulo, Brazil.

Diagnosis of fetal alcohol spectrum disorders (FASD) is challenging and typically requires confirmation of in utero alcohol exposure. Due to the poor reliability of maternal self-reports, biomarkers have emerged to address the problem of obtaining exposure history. A relatively novel method for detecting prenatal alcohol exposure is analysis of meconium for fatty acid ethyl esters (FAEEs), which are non-oxidative ethanol metabolites. Screening newborns using meconium FAEEs may facilitate early diagnosis and intervention in alcohol-affected individuals. The overall objective of this thesis is to further investigate, validate, and assess the clinical utility of meconium FAEE analysis as a screening tool for the identification of neonates at-risk for FASD. This objective was addressed in four separate studies. The first study assessed whether meconium FAEE concentrations can be predictive of ethanol-induced organ injury in fetal sheep, and determined that the levels of these esters could be used to identify fetuses at-risk for organ dysfunction that do not display overt physical signs of ethanol teratogenicity. The second study investigated the effect of delayed meconium collection and contamination with postnatal stool on FAEE analysis, and determined it to be a risk factor for false positive test results. In the third study, maternal willingness to partake in an open meconium screening program was assessed and found to be low enough to diminish the utility of meconium FAEE testing for population-based open screening. Lastly, a systematic review examining the capacity for FAEE synthesis and the enzymology of this non-oxidative metabolic pathway in mammalian organs and tissues revealed that FAEE synthesis is mediated by numerous enzymes and isoenzymes, many of which have other primary physiological functions, and that their contribution to overall FAEE-synthesis may be tissue-specific. Overall, the results of this research provide new information on the benefits, limitations, and utility of meconium FAEE testing as a screening tool for identifying prenatal alcohol exposure − a test that may be of great clinical value in the diagnosis and management of FASD.

Diagnosis of fetal alcohol spectrum disorders (FASD) is challenging and typically requires confirmation of in utero alcohol exposure. Due to the poor reliability of maternal self-reports, biomarkers have emerged to address the problem of obtaining exposure history. A relatively novel method for detecting prenatal alcohol exposure is analysis of meconium for fatty acid ethyl esters (FAEEs), which are non-oxidative ethanol metabolites. Screening newborns using meconium FAEEs may facilitate early diagnosis and intervention in alcohol-affected individuals. The overall objective of this thesis is to further investigate, validate, and assess the clinical utility of meconium FAEE analysis as a screening tool for the identification of neonates at-risk for FASD. This objective was addressed in four separate studies. The first study assessed whether meconium FAEE concentrations can be predictive of ethanol-induced organ injury in fetal sheep, and determined that the levels of these esters could be used to identify fetuses at-risk for organ dysfunction that do not display overt physical signs of ethanol teratogenicity. The second study investigated the effect of delayed meconium collection and contamination with postnatal stool on FAEE analysis, and determined it to be a risk factor for false positive test results. In the third study, maternal willingness to partake in an open meconium screening program was assessed and found to be low enough to diminish the utility of meconium FAEE testing for population-based open screening. Lastly, a systematic review examining the capacity for FAEE synthesis and the enzymology of this non-oxidative metabolic pathway in mammalian organs and tissues revealed that FAEE synthesis is mediated by numerous enzymes and isoenzymes, many of which have other primary physiological functions, and that their contribution to overall FAEE-synthesis may be tissue-specific. Overall, the results of this research provide new information on the benefits, limitations, and utility of meconium FAEE testing as a screening tool for identifying prenatal alcohol exposure − a test that may be of great clinical value in the diagnosis and management of FASD.

O uso de drogas por mulheres em idade reprodutiva é considerado um grave problema de saúde pública mundial. O mecônio é a primeira excreção do recém-nascido e tem sido utilizado como uma matriz alternativa em análises toxicológicas. A extração em fase sólida (SPE) é um método amplamente utilizado para a purificação e concentração dos analitos em amostras biológicas no campo da análise forense. A maioria dos produtos de SPE convencionais requerem volumes relativamente grandes de solventes levando a um custo acrescido por amostra e um aumento no tempo de processamento da amostra. As ponteiras de extração com fase sólida modificada (DPX) foram utilizadas como uma alternativa aos cartuchos SPE tradicionais. A técnica combina eficiência e rapidez no procedimento de extração, com redução significativa no consumo de solvente e na quantidade de amostra. O objetivo desse estudo foi desenvolver e validar um método para a determinação de nicotina, cotinina, cocaína, benzoilecgonina, cocaetileno e éster metil anidroecgonina em amostras de mecônio usando DPX e cromatografia em fase gasosa acoplada à espectrometria de massa (GC/MS). Os resultados da validação indicaram uma extração eficiente, exata e precisa, com recuperação entre 50-98%, exatidão entre 92-106%, precisão intra-ensaio 4-12% e precisão inter-ensaio 6 a 12%. As curvas de calibração foram lineares com valores de R2 superiores a 0,99; os limites de detecção (LOD) variaram entre 2,5-15 ng/g e os limites de quantificação (LOQ) entre 10-20 ng/g. O método DPX-GC/MS mostrou ser eficaz para análise traços de drogas presentes em amostras de mecônio. Após desenvolvimento e validação, o método foi aplicado em 50 amostras de mecônio coletadas no berçário da Maternidade do Complexo Aeroporto (MATER) na cidade de Ribeirão Preto - São Paulo, Brasil. / Drug abuse by pregnant women is considered a serious public health problem worldwide. Meconium is the first excretion in newborns and has been used as an alternative matrix to evaluate in utero drug exposure. Solid phase extraction (SPE) is widely employed to prepare and cleanup samples in the field of forensic analysis. Most SPE products require large volumes of solvent, which culminates in longer sample processing times; increased cost per sample and higher limits of detection. Disposable Pipette Extraction tips (DPX) have been used as an alternative to traditional SPE cartridges. They combine efficient and rapid extraction with reduced solvent consumption. The purpose of this study was to develop and validate a method to determine nicotine, cotinine, cocaine, benzoylecgonine, cocaethylene and methyl ester anhydroecgonine in meconium using DPX and GC/MS. Validation results indicated extraction efficiency, ranged between 50-98%, accuracy 92-106%, intra-assay precision 4-12% and inter-assay precision 6 to 12%. Linear calibration curves resulted in R2 values greater than 0.99; limits of detection ranged from 2.5 - 15 ng/g and the limit of quantitation from 10 - 20 ng/g. The DPX-GC/MS method provided to selectively analyze trace concentrations of drugs in meconium samples. Finally, the developed and validated method was applied to 50 meconium samples collected at the nursery of Maternidade do Complexo Aeroporto (MATER) in the city of Ribeirão Preto - São Paulo, Brazil.

Despite evidence of an association between prenatal acetaminophen exposure and attention deficit hyperactivity disorder (ADHD) in offspring, the causal role of prenatal acetaminophen exposure in child ADHD remains unclear owing to limitations of prior studies. Prior studies have relied on maternal self-report, failed to quantify acetaminophen dose, and lacked mechanistic insight. Chapter 1 formally introduces this topic and provides background information summarizing the high prevalence of ADHD, widespread use of acetaminophen during pregnancy, and potential molecular mechanisms through which the drug may harm fetal development. In Chapter 2, we examined the association between prenatal acetaminophen exposure measured in meconium and ADHD in children aged 6 to 7 years, along with the potential for mediation by functional brain connectivity. Data came from a prospective birth cohort study from the Centre Hospitalier Université de Sherbrooke in Sherbrooke, Québec, Canada. We included 393 eligible children, of whom 345 had meconium samples collected at delivery and information on ADHD diagnosis. Mothers were enrolled from September 25, 2007, to September 10, 2009, at their first prenatal care visit or delivery. Acetaminophen levels were measured in meconium, and physician diagnosis of ADHD was determined at follow-up when children were aged 6 to 7 years or from medical records. Additionally, when children were aged 9 to 11 years, resting-state brain connectivity was assessed with magnetic resonance imaging, and attention problems and hyperactivity were assessed with the Behavioral Assessment System for Children Parent Report Scale. Associations between meconium acetaminophen levels and outcomes were estimated with linear and logistic regressions weighted on the inverse probability of treatment to account for potential confounders. Causal mediation analysis was used to test for mediation of the association between prenatal acetaminophen exposure and hyperactivity by resting-state brain connectivity. Among the 345 children included in the analysis (177 boys [51.3%]; mean [SD] age, 6.58 [0.54] years), acetaminophen was detected in 199 meconium samples (57.7%), and ADHD was diagnosed in 33 children (9.6%). Compared with no acetaminophen, detection of acetaminophen in meconium was associated with increased odds of ADHD (odds ratio [OR], 2.43; 95%CI, 1.41-4.21). A dose-response association was detected; each doubling of exposure increased the odds of ADHD by 10% (OR, 1.10; 95%CI, 1.02-1.19). Children with acetaminophen detected in meconium showed increased negative connectivity between frontoparietal and default mode network nodes to clusters in the sensorimotor cortices, which mediated an indirect effect on increased child hyperactivity (14%; 95%CI, 1%-26%). In Chapter 3, we used data from the same Canadian birth cohort to examine whether prenatal acetaminophen exposure is associated with adverse birth outcomes and/or pregnancy complications, and if birth outcomes may mediate the association of prenatal acetaminophen with child ADHD. This study included 393 children for whom acetaminophen was measured in meconium at delivery. We tested associations of prenatal acetaminophen with birthweight, preterm birth, gestational age, small and large for gestational age, gestational diabetes, preeclampsia, and high blood pressure. Using causal mediation analyses, we assessed whether birth outcomes mediated the association of prenatal acetaminophen with ADHD. We imputed missing data via multiple imputation and used inverse probability weighting to account for confounding and selection bias. Prenatal acetaminophen exposure was associated with decreased birthweight by 136 g (β = −136; 95% CI [−229, −43]), 20% increased weekly hazard of delivery (hazard ratio = 1.20; 95% CI [1.00, 1.43]), and over 60% decreased odds of being born large for gestational age (odds ratio = 0.38; 95% CI [0.20, 0.75]). Prenatal acetaminophen was not associated with small for gestational age, preterm birth, or any pregnancy complications. Causal mediation effects were non-significant for all birth outcomes in both unadjusted and adjusted models, indicating no evidence that birth outcomes linked prenatal acetaminophen exposure with child ADHD. In Chapter 4, we examined the effects of developmental acetaminophen exposure on mouse behavior and frontal cortex gene expression. Although prior studies have investigated neurodevelopmental effects of prenatal acetaminophen exposure in rodents, the results of these studies are not always in agreement. Additionally, no mouse studies of prenatal acetaminophen exposure have investigated offspring attention deficits in behavior tasks specifically designed to measure attention, and no prior rodent studies have utilized ‘omics’ technologies for an untargeted exploration of potential mechanisms. We randomly assigned pregnant mice (starting embryonic day 4-10) to receive acetaminophen (150 mg/kg/day) or vehicle control through postnatal day 14. We employed a battery of behavior tests for 111 mouse offspring, including pup ultrasonic vocalizations, elevated plus maze, open field test, CatWalk, pre-pulse inhibition, and 5-choice serial reaction time task. Frontal cortex was collected at birth from 24 pups for RNA-sequencing. Developmental acetaminophen treatment resulted in increased pup vocalizations after separation from the litter, as well as decreased ambulation and vertical rearings in the open field task among male but not female offspring. Acetaminophen treatment was also associated with altered frontal cortex gene expression relating to glutathione and cytochrome p450 metabolism, DNA damage, and the endocrine and immune systems. Together with the multitude of other cohort studies showing adverse neurodevelopment associated with prenatal acetaminophen exposure, this work suggests caution should be used in administering acetaminophen during pregnancy. In humans, we found that prenatal acetaminophen exposure was associated with child ADHD, altered resting-state brain connectivity, and adverse birth outcomes. Furthermore, our results suggest altered brain connectivity as a potential underlying mechanism linking prenatal acetaminophen use with child hyperactivity. While adverse birth outcomes such as preterm birth and reduced birthweight are known to be associated with ADHD, we found no evidence for mediation by birth outcomes of the association between prenatal acetaminophen exposure and ADHD. In mice, we found that developmental acetaminophen treatment resulted in elevated anxiety-like behaviors in male offspring, as well as gene expression changes in the frontal cortex. Future studies are needed to explore whether the altered molecular pathways revealed by RNA-sequencing directly link acetaminophen exposure with offspring behavior changes.

O tabaco é uma das principais drogas consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, têm representado uma grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de fumar durante a gestação serem bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. A exposição fetal aos constituintes do tabaco tem sido associada com aumento do risco de aborto espontâneo e nascimentos prematuros, incidência de recém-nascidos com baixo peso, síndrome de morte súbita infantil e desordens cognitivas e neurocomportamentais. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado tabaco durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal aos constituintes do tabaco. Dentre os marcadores utilizados estão a nicotina e o seu principal produto de biotransformação, a cotinina, que podem ser detectados em amostras de mecônio de recém-nascidos. No presente estudo, um método analítico foi desenvolvido visando à detecção desses marcadores em amostras de mecônio. Foi considerada neste projeto a técnica de extração acelerada por solvente (ASE) por ser uma técnica promissora para o preparo de amostras sólidas e/ou semi-sólidas. Os analitos foram identificados por cromatografia em fase-gasosa com detector de nitrogênio e fósforo (GC/NPD). Os limites de detecção foram de 3 ng/g e 30 ng/g e os de quantificação foram de 5 ng/g e 40 ng/g, para cotinina e nicotina respectivamente, e apresentaram boa linearidade na faixa de concentração estudada (5-500 ng/g), com coeficiente de correlação (r2) maior que 0,98. A precisão intra-ensaio, determinada pelo coeficiente de variação do método (CV%) foi menor que 15% e a precisão interensaio foram menor ou igual a 20% para nicotina e cotinina. A recuperação média foi de 77%. O método desenvolvido demonstrou ser rápido, preciso, prático e sensível e com uso de menores volumes de solventes orgânicos do que outros métodos descritos na literatura. O método desenvolvido e validado foi aplicado em amostras de mecônio de neonatos com suspeita ou não de exposição fetal aos constituintes do tabaco. / Tobacco is one of the main drugs consumed worldwide and its use by women of reproductive age in particular has played a major concern among experts and society in general. Despite the adverse effects associated with smoking during pregnancy are well documented, it is known that a significant proportion of pregnant women have difficulties to quit the habit. Fetal exposure to tobacco constituents has been associated with increased risk of spontaneous abortion (miscarriage) and premature births, incidence of newborns with low birth weight, sudden infant death syndrome and neurobehavioral and cognitive disorders. However, due to guilt and fear of punitive actions, women rarely admit to have used tobacco during pregnancy. As a result, a series of biological markers have been studied to diagnose fetal exposure to tobacco constituents. Nicotine and cotinine are some of these biomarkers which can be detected in meconium samples from newborns. In this study, an analytical method was developed to detect these biomarkers in meconium samples. Accelerated solvent extraction (ASE), a promising technique for solid or semi-solid sample preparation, was considered in this work the that is considered a. The analytes were identified by gas-chromatography with nitrogen phosphorus detector (GC / NPD). The limits of detection (LOD) were 3 ng/g and 30 ng/g and the limits of quantification (LOQ) were 5 ng/g and 40 ng/g for cotinine and nicotine, respectively. The method showed good linearity in the concentration range studied (5-500 ng/g), with a coefficient of correlation (r2) greater than 0.98. The intraday precision, determined by the coefficient of variation (CV %) was less than 15% and the interday precision was less or equal than 20% for nicotine and cotinine. The average recovery was 77%. The method proved to be fast, accurate, practical and sensitive and smaller volumes of organic solvents are necessary, compared to other methods published in the scientific literature. The developed and validated method was applied to meconium samples of suspected and nonsuspected neonates of having been exposed to tobacco constituents during gestation.

O consumo de cocaína e crack no Brasil é um problema de saúde pública, ainda mais grave quando realizado por gestantes, que colocam em risco a sua vida e a do feto. A identificação dessas substâncias através de cromatografia gasosa acoplada à espectrometria de massas e utilizando como matriz biológica o mecônio, é uma técnica eficiente para detecção da exposição fetal à cocaína. O mecônio apresenta algumas vantagens em relação às outras matrizes biológicas como uma ampla janela de detecção dos analitos e de fácil coleta por ser não-invasiva. Diante disso, o objetivo do presente estudo foi desenvolver e validar uma metodologia de preparo do mecônio para identificação de cocaína, benzoilecgonina, cocaetileno e o éster metilanidroecgonina em seus extratos, usando a cromatografia gasosa acoplada a espectrometria de massas (CG-EM). Os analitos foram inicialmente extraídos com metanol, sendo posteriormente, purificados usando cartuchos de extração em fase sólida do tipo Bond Elut Certify I. A determinação desses analitos foi realizada usando um CG-EM do tipo íon trap, no modo full scan de detecção. O método foi validado, segundo critérios estabelecidos pela ANVISA, na faixa de linearidade de 20 a 1000 ng/g para a cocaína e o cocaetileno; 40 a 1500 ng/g para a benzoilecgonina e 60 a 1500 ng/g para o éster metilanidroecgonina, usando 0,5 g de mecônio por análise. A resposta do detector apresentou-se linear na faixa estudada e o limite de detecção encontrado foi de 10ng/g para a cocaína; 20ng/g para o cocaetileno; 30ng/g para a benzoilecgonina e 40 ng/g para o éster metilanidroecgonina. O coeficiente de variação intra-ensaio variou de 3,01% a 10,15% e o inter-ensaio variou entre 5,31% a 11,12%; a exatidão variou entre 91,47% e 105,31%. A recuperação encontrada foi superior a 56,30%. A especificidade foi determinada para os seguintes interferentes: AAS (ácido acetilsalicílico), alprazolam, anfetamina, cafeína, dipirona, efedrina, fenilefrina, fluoxetina, metoclopramida, nicotina, sulfato ferroso e THC (tetraidrocanabinol). Após o término da validação, o método foi aplicado em amostras de mecônio coletadas de recém-nascidos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HCFMRP USP). A coleta destas amostras foi realizada após a autorização das mães em participarem da pesquisa, através da assinatura do Termo de Consentimento Livre e Esclarecido, e após preencherem um questionário sobre consumo de drogas. O método desenvolvido e validado mostrou ser eficiente na identificação de cocaína, metabólitos e derivados em mecônio. / Cocaine and crack use is an important public health problem in Brazil, even though when this is made by pregnants, risking the babies lives besides their own. The identification of such substances through gas chromatograph-mass spectrometry using meconium as biological matrix is highly efficient on detecting fetal exposure to cocaine. The meconium presents some advantages in comparison with other matrices such as analite wide window detection and collection facilities by non-invasive methods. The purpose of this study was to develop and validate a method for meconium sample preparation for a gas chromatography-mass spectrometry (GC-MS) confirmation of meconium extracts for cocaine, benzoylecgonine, cocaethylene and anhydroecgonine methyl ester. The analytes were initially extracted from the matrix by methanol. Then, a solid-phase extraction with Bond Elut Certify I cartridges was applied. Analytes were determined in a GC-MS ion trap, full scan mode. The method was validated in the range of 20 -1000 ng/g for cocaine and cocaethylene; 40 -1500 ng/g for benzoylecgonine and 60 -1500 ng/g for anhydroecgonine methyl ester, using 0.5 g of meconium per assay. The detector response was linear in the studied range and limit of detection were found to be 10ng/g to cocaine; 20ng/g to cocaethylene; 30ng/g to benzoylecgonine and 40 ng/g to anhydroecgonine methyl ester. Intra-batch coefficients of variation oscillated between 3.01% and 10.15% and inter-batch oscillated between 5.31% and 11.12%; accuracy were in range 91.47% - 105.31%. The recoveries were higher than 56.30%. Selectiviy was determined for these interferents: AAS (acetylsalicylic acid), alprazolam, amphetamine, caffeine, dipyrone, ephedrine, phenylephrine, fluoxetine, metoclopramide, nicotine, iron sulfate and THC (tetrahydrocanabynol). Finally, the method was applied to analysis of meconium collected from newborns in the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HCFMRP USP). The sample collections were made after mothers´s authorization, and after they signed a term and answered a self-report about use of drugs. The developed and validated method was efficient on identification of cocaine, metabolites and derivatives in meconium.

O consumo de drogas de abuso é grave problema de saúde pública em todo o mundo. No Brasil observa-se aumento no número de usuárias em idade fértil, levando a crescente preocupação com relação à exposição fetal. Efeitos deletérios como diminuição do peso e crescimento fetal, parto prematuro, déficits neurológicos e comportamentais estão associados ao uso de cocaína e cannabis durante a gestação. Portanto, é importante obter informações acerca do consumo de drogas durante a gravidez, permitindo intervenções médicas e psicológicas adequadas. Os relatos fornecidos pelas gestantes são relevantes, porém muitas vezes resultam em dados subestimados devido à omissão de informações por medo de ações punitivas. Desta forma, a confirmação por meio de análises toxicológicas em amostras biológicas se faz necessária. O mecônio, primeiras fezes do recém-nascido, tem sido proposto como matriz adequada para avaliação da exposição fetal, pois apresenta coleta não invasiva, fácil obtenção e fornece informações de longo prazo (2º e 3º trimestre de gestação). Entretanto, é uma matriz complexa, exigindo diversas etapas de purificação para posterior análise. No presente trabalho, métodos analíticos foram desenvolvidos visando à detecção dos biomarcadores da exposição fetal à cocaína e ao tetraidrocanabinol em amostras de mecônio por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Para ambos os métodos, a extração acelerada por solvente (ASE) foi utilizada para o isolamento dos analitos de interesse de amostras de mecônio, já que esta apresenta vantagens frente às técnicas convencionais, devido sua maior eficiência e menor manipulação da amostra. Associada à ASE, a extração em fase sólida (SPE) foi empregada para purificação e concentração dos analitos de interesse. Na etapa de desenvolvimento, inicialmente foram estabelecidos os procedimentos de derivatização e as condições cromatográficas a serem empregadas nas análises. Posteriormente, realizou-se a otimização dos procedimentos empregados na ASE, através de análise de superfície de resposta. Os métodos foram validados de acordo com o preconizado por referências internacionais, estabelecendo-se os limites de detecção e quantificação, recuperação, linearidade, precisão intra e interensaio e exatidão. O método para detecção dos biomarcadores da cocaína foi aplicado em 342 amostras de mecônio, provenientes do Hospital Universitário da Universidade de São Paulo (HU-USP). Destas, 19 (5,6%) apresentaram resultado positivo para um ou mais biomarcadores da exposição fetal à cocaína. Além disto, foi observada redução estatisticamente significante do peso ao nascimento, comprimento e perímetro cefálico entre os recém-nascidos expostos à cocaína durante a gestação. O método para a detecção da exposição fetal ao tetraidrocanabinol foi aplicado em 6 amostras positivas, obtidas do HU-USP, mostrando a aplicabilidade da técnica desenvolvida. / The use of illicit drugs is a relevant public health problem in the world. In Brazil, the number of women users in fertile age is increasing, which leads to a growing concern regarding fetal drug exposure. Adverse outcomes such as low birth weight, intra-uterine growth restriction, preterm birth, neurobehavioral and developmental deficits are associated with cocaine and cannabis use during pregnancy. Consequently, it would be important to obtain data related to drug misuse during gestation with the aim to plan medical and psychological interventions. Self-report drug use by pregnant women is often inaccurate due to feelings of guilt or fear of punitive actions. Therefore, confirmation by toxicological analysis in biological matrices must be accomplished. Meconium, the first stool of the newborn, has been proposed as a proper matrix to evaluate fetal exposure because it is collected by an easy and non-invasive way and enables the achievement of long-term information regarding fetal exposure. However, meconium is a complex matrix, which requires extensive sample cleaning previously to the analytes identification. In the present research, analytical methods were developed aiming the determination of cocaine and tetrahydrocannabinol biomarkers in meconium samples through gas chromatography-mass spectrometry (GC-MS). Accelerated solvent extraction (ASE) was used for analytes isolation due to its advantages over conventional techniques, such as greater extraction efficiency and minor sample handling. In order to achieve proper analytes selectivity and detectability, solid phase extraction (SPE) was employed for post-extraction clean-up. Initially, the derivatization procedure and chromatographic parameters were established for the development of the methods. Afterwards, ASE procedure was optimized through response surface methodology. The analytical methods were validated in accordance to international references. Limits of detection and quantification, recovery, linearity, precision and accuracy were obtained. The developed method for cocaine was applied in 342 meconium samples collected from the University Hospital of University of São Paulo (HU-USP). Among them, 19 (5.6%) showed positivity result for cocaine biomarkers. Additionally, newborns from mothers exposed to cocaine exhibited statistical significant lower birth weight, length and head circumference when compared with newborns from non-consumer mothers. For the method of tetrahydrocannabinol, applicability and importance was demonstrated by analyzing 6 positive samples from HU-USP.

O consumo de drogas de abuso é grave problema de saúde pública em todo o mundo. No Brasil observa-se aumento no número de usuárias em idade fértil, levando a crescente preocupação com relação à exposição fetal. Efeitos deletérios como diminuição do peso e crescimento fetal, parto prematuro, déficits neurológicos e comportamentais estão associados ao uso de cocaína e cannabis durante a gestação. Portanto, é importante obter informações acerca do consumo de drogas durante a gravidez, permitindo intervenções médicas e psicológicas adequadas. Os relatos fornecidos pelas gestantes são relevantes, porém muitas vezes resultam em dados subestimados devido à omissão de informações por medo de ações punitivas. Desta forma, a confirmação por meio de análises toxicológicas em amostras biológicas se faz necessária. O mecônio, primeiras fezes do recém-nascido, tem sido proposto como matriz adequada para avaliação da exposição fetal, pois apresenta coleta não invasiva, fácil obtenção e fornece informações de longo prazo (2º e 3º trimestre de gestação). Entretanto, é uma matriz complexa, exigindo diversas etapas de purificação para posterior análise. No presente trabalho, métodos analíticos foram desenvolvidos visando à detecção dos biomarcadores da exposição fetal à cocaína e ao tetraidrocanabinol em amostras de mecônio por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Para ambos os métodos, a extração acelerada por solvente (ASE) foi utilizada para o isolamento dos analitos de interesse de amostras de mecônio, já que esta apresenta vantagens frente às técnicas convencionais, devido sua maior eficiência e menor manipulação da amostra. Associada à ASE, a extração em fase sólida (SPE) foi empregada para purificação e concentração dos analitos de interesse. Na etapa de desenvolvimento, inicialmente foram estabelecidos os procedimentos de derivatização e as condições cromatográficas a serem empregadas nas análises. Posteriormente, realizou-se a otimização dos procedimentos empregados na ASE, através de análise de superfície de resposta. Os métodos foram validados de acordo com o preconizado por referências internacionais, estabelecendo-se os limites de detecção e quantificação, recuperação, linearidade, precisão intra e interensaio e exatidão. O método para detecção dos biomarcadores da cocaína foi aplicado em 342 amostras de mecônio, provenientes do Hospital Universitário da Universidade de São Paulo (HU-USP). Destas, 19 (5,6%) apresentaram resultado positivo para um ou mais biomarcadores da exposição fetal à cocaína. Além disto, foi observada redução estatisticamente significante do peso ao nascimento, comprimento e perímetro cefálico entre os recém-nascidos expostos à cocaína durante a gestação. O método para a detecção da exposição fetal ao tetraidrocanabinol foi aplicado em 6 amostras positivas, obtidas do HU-USP, mostrando a aplicabilidade da técnica desenvolvida. / The use of illicit drugs is a relevant public health problem in the world. In Brazil, the number of women users in fertile age is increasing, which leads to a growing concern regarding fetal drug exposure. Adverse outcomes such as low birth weight, intra-uterine growth restriction, preterm birth, neurobehavioral and developmental deficits are associated with cocaine and cannabis use during pregnancy. Consequently, it would be important to obtain data related to drug misuse during gestation with the aim to plan medical and psychological interventions. Self-report drug use by pregnant women is often inaccurate due to feelings of guilt or fear of punitive actions. Therefore, confirmation by toxicological analysis in biological matrices must be accomplished. Meconium, the first stool of the newborn, has been proposed as a proper matrix to evaluate fetal exposure because it is collected by an easy and non-invasive way and enables the achievement of long-term information regarding fetal exposure. However, meconium is a complex matrix, which requires extensive sample cleaning previously to the analytes identification. In the present research, analytical methods were developed aiming the determination of cocaine and tetrahydrocannabinol biomarkers in meconium samples through gas chromatography-mass spectrometry (GC-MS). Accelerated solvent extraction (ASE) was used for analytes isolation due to its advantages over conventional techniques, such as greater extraction efficiency and minor sample handling. In order to achieve proper analytes selectivity and detectability, solid phase extraction (SPE) was employed for post-extraction clean-up. Initially, the derivatization procedure and chromatographic parameters were established for the development of the methods. Afterwards, ASE procedure was optimized through response surface methodology. The analytical methods were validated in accordance to international references. Limits of detection and quantification, recovery, linearity, precision and accuracy were obtained. The developed method for cocaine was applied in 342 meconium samples collected from the University Hospital of University of São Paulo (HU-USP). Among them, 19 (5.6%) showed positivity result for cocaine biomarkers. Additionally, newborns from mothers exposed to cocaine exhibited statistical significant lower birth weight, length and head circumference when compared with newborns from non-consumer mothers. For the method of tetrahydrocannabinol, applicability and importance was demonstrated by analyzing 6 positive samples from HU-USP.

O tabaco é uma das principais drogas consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, têm representado uma grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de fumar durante a gestação serem bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. A exposição fetal aos constituintes do tabaco tem sido associada com aumento do risco de aborto espontâneo e nascimentos prematuros, incidência de recém-nascidos com baixo peso, síndrome de morte súbita infantil e desordens cognitivas e neurocomportamentais. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado tabaco durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal aos constituintes do tabaco. Dentre os marcadores utilizados estão a nicotina e o seu principal produto de biotransformação, a cotinina, que podem ser detectados em amostras de mecônio de recém-nascidos. No presente estudo, um método analítico foi desenvolvido visando à detecção desses marcadores em amostras de mecônio. Foi considerada neste projeto a técnica de extração acelerada por solvente (ASE) por ser uma técnica promissora para o preparo de amostras sólidas e/ou semi-sólidas. Os analitos foram identificados por cromatografia em fase-gasosa com detector de nitrogênio e fósforo (GC/NPD). Os limites de detecção foram de 3 ng/g e 30 ng/g e os de quantificação foram de 5 ng/g e 40 ng/g, para cotinina e nicotina respectivamente, e apresentaram boa linearidade na faixa de concentração estudada (5-500 ng/g), com coeficiente de correlação (r2) maior que 0,98. A precisão intra-ensaio, determinada pelo coeficiente de variação do método (CV%) foi menor que 15% e a precisão interensaio foram menor ou igual a 20% para nicotina e cotinina. A recuperação média foi de 77%. O método desenvolvido demonstrou ser rápido, preciso, prático e sensível e com uso de menores volumes de solventes orgânicos do que outros métodos descritos na literatura. O método desenvolvido e validado foi aplicado em amostras de mecônio de neonatos com suspeita ou não de exposição fetal aos constituintes do tabaco. / Tobacco is one of the main drugs consumed worldwide and its use by women of reproductive age in particular has played a major concern among experts and society in general. Despite the adverse effects associated with smoking during pregnancy are well documented, it is known that a significant proportion of pregnant women have difficulties to quit the habit. Fetal exposure to tobacco constituents has been associated with increased risk of spontaneous abortion (miscarriage) and premature births, incidence of newborns with low birth weight, sudden infant death syndrome and neurobehavioral and cognitive disorders. However, due to guilt and fear of punitive actions, women rarely admit to have used tobacco during pregnancy. As a result, a series of biological markers have been studied to diagnose fetal exposure to tobacco constituents. Nicotine and cotinine are some of these biomarkers which can be detected in meconium samples from newborns. In this study, an analytical method was developed to detect these biomarkers in meconium samples. Accelerated solvent extraction (ASE), a promising technique for solid or semi-solid sample preparation, was considered in this work the that is considered a. The analytes were identified by gas-chromatography with nitrogen phosphorus detector (GC / NPD). The limits of detection (LOD) were 3 ng/g and 30 ng/g and the limits of quantification (LOQ) were 5 ng/g and 40 ng/g for cotinine and nicotine, respectively. The method showed good linearity in the concentration range studied (5-500 ng/g), with a coefficient of correlation (r2) greater than 0.98. The intraday precision, determined by the coefficient of variation (CV %) was less than 15% and the interday precision was less or equal than 20% for nicotine and cotinine. The average recovery was 77%. The method proved to be fast, accurate, practical and sensitive and smaller volumes of organic solvents are necessary, compared to other methods published in the scientific literature. The developed and validated method was applied to meconium samples of suspected and nonsuspected neonates of having been exposed to tobacco constituents during gestation.