Application of Bar Adsorptive Microextraction for the Determination of Levels of Tricyclic Antidepressants in Urine Samples.

This work entailed the development, optimization, validation, and application of a novel analytical approach, using the bar adsorptive microextraction technique (BAµE), for the determination of the six most common tricyclic antidepressants (TCAs; amitriptyline, mianserin, trimipramine, imipramine, mirtazapine and dosulepin) in urine matrices. To achieve this goal, we employed, for the first time, new generation microextraction devices coated with convenient sorbent phases, polymers and novel activated carbons prepared from biomaterial waste, in combination with large-volume-injection gas chromatography-mass spectrometry operating in selected-ion monitoring mode (LVI-GC-MS(SIM)). Preliminary assays on sorbent coatings, showed that the polymeric phases present a much more effective performance, as the tested biosorbents exhibited low efficiency for application in microextraction techniques. By using BAµE coated with C18 polymer, under optimized experimental conditions, the detection limits achieved for the six TCAs ranged from 0.2 to 1.6 µg L-1 and, weighted linear regressions resulted in remarkable linearity (r2 > 0.9960) between 10.0 and 1000.0 µg L-1. The developed analytical methodology (BAµE(C18)/LVI-GC-MS(SIM)) provided suitable matrix effects (90.2-112.9%, RSD ≤ 13.9%), high recovery yields (92.3-111.5%, RSD ≤ 12.3%) and a remarkable overall process efficiency (ranging from 84.9% to 124.3%, RSD ≤ 13.9%). The developed and validated methodology was successfully applied for screening the six TCAs in real urine matrices. The proposed analytical methodology proved to be an eco-user-friendly approach to monitor trace levels of TCAs in complex urine matrices and an outstanding analytical alternative in comparison with other microextraction-based techniques.

Application of Microextraction-Based Techniques for Screening-Controlled Drugs in Forensic Context-A Review.

The analysis of controlled drugs in forensic matrices, i.e., urine, blood, plasma, saliva, and hair, is one of the current hot topics in the clinical and toxicological context. The use of microextraction-based approaches has gained considerable notoriety, mainly due to the great simplicity, cost-benefit, and environmental sustainability. For this reason, the application of these innovative techniques has become more relevant than ever in programs for monitoring priority substances such as the main illicit drugs, e.g., opioids, stimulants, cannabinoids, hallucinogens, dissociative drugs, and related compounds. The present contribution aims to make a comprehensive review on the state-of-the art advantages and future trends on the application of microextraction-based techniques for screening-controlled drugs in the forensic context.

Bar Adsorptive Microextraction Coated with Carbon-based Phase Mixtures for Performance-Enhancement to Monitor Selected Benzotriazoles, Benzothiazoles, and Benzenesulfonamides in Environmental Water Matrices.

: In the present work we propose, for the first time, bar adsorptive microextraction coated with carbon-based phase mixtures, followed by microliquid desorption and high performance liquid chromatography-diode array detection (BAµE-µLD/HPLC-DAD) analysis, to enhance the performance of the determination of traces of benzotriazoles (BTRs), benzothiazoles (BTs), and benzenesulfonamide derivatives (BSDs) in environmental water matrices. Assessing six carbon-based sorbents (CA1, CN1, B test EUR, SX PLUS, SX 1, and R) with different selectivity properties allowed us to tailor the best phase mixture (R, 12.5%/CN1, 87.5%) that has convenient porosity, texture, and surface chemistry (pHPZC,mix ~6.5) for trace analysis of benzenesulfonamide, 1-hydroxybenzotriazole, 1H-benzotriazole, 5-methyl-1H-benzotriazole, benzothiazole, and 1,3-benzothiazol-2-ol chemicals in aqueous media. Optimized experimental conditions provided average recoveries ranging from 37.9% to 59.2%, appropriate linear dynamic ranges (5.0 to 120.0 µg L-1; r2 ≥ 0.9964), limits of detection between 1.0 and 1.4 µg L-1, and good precisions (relative standard deviation (RSD) ≤ 9.3%). The proposed methodology (BAµE(R, 12.5%/CN1, 87.5%)-µLD/HPLC-DAD) also proved to be a suitable sorption-based static microextraction alternative to monitor traces of BTRs, BTs, and BSDs in rain, waste, tap, and estuarine water samples. From the data obtained, the proposed approach showed that the BAµE technique with the addition of lab-made devices allows users to adapt the technique to use sorbents or mixtures of sorbents with the best selectivity characteristics whenever distinct classes of target analytes occur simultaneously in the same application.

A Fast and Validated High Throughput Bar Adsorptive Microextraction (HT-BAµE) Method for the Determination of Ketamine and Norketamine in Urine Samples.

We developed, optimized and validated a fast analytical cycle using high throughput bar adsorptive microextraction and microliquid desorption (HT-BAµE-µLD) for the extraction and desorption of ketamine and norketamine in up to 100 urine samples simultaneously, resulting in an assay time of only 0.45 min/sample. The identification and quantification were carried out using large volume injection-gas chromatography-mass spectrometry operating in the selected ion monitoring mode (LVI-GC-MS(SIM)). Several parameters that could influencing HT-BAµE were assayed and optimized in order to maximize the recovery yields of ketamine and norketamine from aqueous media. These included sorbent selectivity, desorption solvent and time, as well as shaking rate, microextraction time, matrix pH, ionic strength and polarity. Under optimized experimental conditions, suitable sensitivity (1.0 µg L-1), accuracy (85.5-112.1%), precision (≤15%) and recovery yields (84.9-105.0%) were achieved. Compared to existing methods, the herein described analytical cycle is much faster, environmentally friendly and cost-effective for the quantification of ketamine and norketamine in urine samples. To our knowledge, this is the first work that employs a high throughput based microextraction approach for the simultaneous extraction and subsequent desorption of ketamine and norketamine in up to 100 urine samples simultaneously.

The RET p.G533C mutation confers predisposition to multiple endocrine neoplasia type 2A in a Brazilian kindred and is able to induce a malignant phenotype in vitro and in vivo.

BACKGROUND: We have previously described a p.G533C substitution in the rearranged during transfection (RET) oncogene in a large family with medullary thyroid carcinoma. Here, we explore the functional transforming potential of RET p.G533C mutation. METHODS: Plasmids expressing RET mutants (p.G533C and p.C634Y) and RET wild type were stable transfected into a rat thyroid cell line (PCCL3). Biological and biochemical effects of RET p.G533C were investigated both in vitro and in vivo. Moreover, we report the first case of pheochromocytoma among the RET p.G533C-carriers in this Brazilian family and explore the RET mutational status in DNA isolated from pheochromocytoma. RESULTS: Ectopic expression of RET p.G533C and p.C634Y activates RET/MAPK/ERK pathway at similar levels and significantly increased cell proliferation, compared with RET wild type. We additionally show that p.G533C increased cell viability, anchorage-independent growth, and micronuclei formation while reducing apoptosis, hallmarks of the malignant phenotype. RET p.G533C down-regulates the expression of thyroid specific genes in PCCL3. Moreover, RET p.G533C-expressing cells were able to induce liver metastasis in nude mice. Finally, we described two novel RET variants (G548V and S556T) in the DNA isolated from pheochromocytoma while they were absent in the DNA isolated from blood. CONCLUSIONS: Our in vitro and in vivo analysis indicates that this mutation confers a malignant phenotype to PCCL3 cells. These findings, in association with the report of first case of pheochromocytoma in the Brazilian kindred, suggest that this noncysteine mutation may be more aggressive than was initially considered.

Riscos isolados e agregados de anemia em crianças frequentadoras de berçários de creches / Isolated and combined risks for anemia in children attending the nurseries of daycare centers

OBJETIVO: Identificar e quantificar os fatores de risco isolados e agregados de anemia, proporcionando visão ampliada quanto à probabilidade da sua ocorrência. MÉTODOS: Estudo transversal com 482 crianças, entre 4 e 29 meses de idade, frequentadoras de berçários de creches públicas e filantrópicas do município de São Paulo (SP), que participaram de dois inquéritos (2004 e 2007). Foram realizadas entrevistas com as mães, coleta de sangue por punção digital e antropometria. Considerou-se anemia, hemoglobina inferior a 11 g/dL. Foi ajustado modelo de regressão logística não-condicional para fatores de risco de anemia, considerando-se estatisticamente significantes associações com p < 0,05. Para definição das probabilidades pós-teste, foram utilizados os cálculos das chances pós-teste e razões de verossimilhança. Os pacotes estatísticos utilizados foram o Epi-InfoTM 2000 e o Stata 10.0. RESULTADOS: A prevalência de anemia foi de 43,6 por cento (IC95 por cento - 39,1 a 48,1 por cento). O modelo logístico final incluiu cinco variáveis: idade materna inferior a 28 anos (OR = 1,50; p = 0,041), renda per capita inferior a meio salário mínimo (OR = 1,56; p = 0,029), aleitamento materno exclusivo inferior a 2 meses (OR = 1,71; p = 0,009), perda de escore z peso/idade do nascimento ao inquérito (OR = 1,47; p = 0,050) e idade inferior a 17 meses (OR = 2,44; p < 0,001). As probabilidades pós-teste nas associações de fatores de risco de anemia isolados e agregados apresentaram-se entre 54,5 e 100 por cento. CONCLUSÕES: A probabilidade de anemia aumentou quanto mais agregados estiveram os cinco fatores de risco identificados. Esse cálculo disponibiliza instrumento simples e rápido de suspeição de anemia em crianças em triagem clínica ou populacional.

OBJECTIVE: To identify and quantify isolated and combined risk factors for anemia, providing a comprehensive view of the likelihood of its occurrence. METHODS: Cross-sectional study with 482 children aged 4 to 29 months attending the nurseries of philanthropic and public daycare centers in the city of São Paulo, Brazil, who participated in two surveys (2004 and 2007). Mothers were interviewed, blood was collected using digital puncture, and anthropometry was performed. Anemia was characterized by hemoglobin levels below 11 g/dL. Unconditional logistic regression was adjusted for anemia risk factors. A value of p < 0.05 indicated statistically significant associations. Post-test odds and likelihood ratios were calculated to define post-test probabilities. Epi-InfoTM 2000 and Stata 10.0 software packages were used for statistical analysis. RESULTS: Prevalence of anemia was 43.6 percent (95 percentCI 39.1-48.1). The final logistic model included five categorical variables: mother's age less than 28 years (OR = 1.50; p = 0.041), per capita income below half a minimum wage (OR = 1.56; p = 0.029), exclusive breastfeeding less than 2 months (OR = 1.71; p = 0.009), decrease in weight/age z score from birth to survey (OR = 1.47; p = 0.050), and age less than 17 months (OR = 2.44; p < 0,001). Post-test probabilities in the associations of isolated and combined risk factors for anemia ranged from 54.5 to 100 percent. CONCLUSIONS: The probability of anemia progressively increased as the identified risk factors were added. This calculation provides a simple and rapid tool for suspicion of anemia in children both in clinical practice and population screening.

Isolated and combined risks for anemia in children attending the nurseries of daycare centers.

OBJECTIVES: To identify and quantify isolated and combined risk factors for anemia, providing a comprehensive view of the likelihood of its occurrence. METHODS: Cross-sectional study with 482 children aged 4 to 29 months attending the nurseries of philanthropic and public daycare centers in the city of São Paulo, Brazil, who participated in two surveys (2004 and 2007). Mothers were interviewed, blood was collected using digital puncture, and anthropometry was performed. Anemia was characterized by hemoglobin levels below 11 g/dL. Unconditional logistic regression was adjusted for anemia risk factors. A value of p < or = 0.05 indicated statistically significant associations. Post-test odds and likelihood ratios were calculated to define post-test probabilities. Epi-Info 2000 and Stata 10.0 software packages were used for statistical analysis. RESULTS: Prevalence of anemia was 43.6% (95%CI 39.1-48.1). The final logistic model included five categorical variables: mother's age less than 28 years (OR = 1.50; p = 0.041), per capita income below half a minimum wage (OR = 1.56; p = 0.029), exclusive breastfeeding less than 2 months (OR = 1.71; p = 0.009), decrease in weight/age z score from birth to survey (OR = 1.47; p = 0.050), and age less than 17 months (OR = 2.44; p < 0,001). Post-test probabilities in the associations of isolated and combined risk factors for anemia ranged from 54.5 to 100%. CONCLUSIONS: The probability of anemia progressively increased as the identified risk factors were added. This calculation provides a simple and rapid tool for suspicion of anemia in children both in clinical practice and population screening.

Early diagnosis of multiple endocrine neoplasia type 2B: a challenge for physicians / Diagnóstico precoce de neoplasia endócrina múltipla tipo 2B: um desafio para os médicos

BACKGROUND: The hereditary form of medullary thyroid carcinoma may occur isolated as a familial medullary thyroid carcinoma (FMTC) or as part of Multiple Endocrine Neoplasia 2A (MEN2A) and 2B (MEN2B). MEN2B is a rare syndrome, its phenotype may usually, but not always, be noted by the physician. In the infant none of the MEN2B characteristics are present, except by early gastrointestinal dysfunction caused by intestinal neuromas. When available, genetic analysis confirms the diagnosis and guides pre-operative evaluation and extent of surgery. Here we report four cases of MEN2B in which the late diagnosis had a significant impact in clinical evolution and, potentially, in overall survival...

A forma hereditária do carcinoma medular da tiróide pode ocorrer de modo isolado, o carcinoma medular da tiróide familiar (FMTC), ou como parte das neoplasias endócrinas múltiplas tipo 2A (MEN2A) e 2B (MEN2B). MEN2B é uma síndrome rara e seu fenótipo é usualmente, mas nem sempre, notado pelo médico. Na infância, nenhuma das características de MEN2B estão presentes, exceto pela disfunção gastrintestinal precoce, causada pelos neuromas intestinais. Quando disponível, a análise genética confirma o diagnóstico e orienta a avaliação pré-operatória e extensão da cirurgia. Neste artigo, apresentamos quatro casos de MEN2B, nos quais o diagnóstico tardio teve impacto significativo na evolução clínica e, potencialmente, na mortalidade em geral...

Early diagnosis of multiple endocrine neoplasia type 2B: a challenge for physicians.

BACKGROUND: The hereditary form of medullary thyroid carcinoma may occur isolated as a familial medullary thyroid carcinoma (FMTC) or as part of Multiple Endocrine Neoplasia 2A (MEN2A) and 2B (MEN2B). MEN2B is a rare syndrome, its phenotype may usually, but not always, be noted by the physician. In the infant none of the MEN2B characteristics are present, except by early gastrointestinal dysfunction caused by intestinal neuromas. When available, genetic analysis confirms the diagnosis and guides pre-operative evaluation and extent of surgery. Here we report four cases of MEN2B in which the late diagnosis had a significant impact in clinical evolution and, potentially, in overall survival. CASE REPORT: We report four cases, 2 men and 2 women, with differences in their phenotypes and with a late diagnosis. The first case has a history of severe gastrointestinal obstruction requiring a surgery intervention two days after his birth. The second told had nodules in the oral mucosa and constipation since childhood. The third case referred a history of constipation from birth until 5 months of life. The fourth has had a history of chronic constipation since childhood. DISCUSSION: New concepts have emerged since the RET oncogene was identified in 1993 as the responsible gene for hereditary medullary thyroid carcinoma. The majority of MEN2B individuals have M918T mutation in the exon 16 of RET, with a few cases having a mutation A883F or the association of V804M with E805K, Y806C or S904C mutations. The consensus classifies the RET mutation in codon 918 as of highest risk and recommends total thyroidectomy and central lymph node dissection until 6 months after birth. A fast and precise diagnosis is essential to reach these goals. The identification of early manifestations such as intestinal ganglioneuromatosis and oral mucosal neuromas should prompt the physician to initiate an investigation for multiple endocrine neoplasia type 2B. CONCLUSION: The diagnosis of MEN2B is very important to allow appropriate investigation of associated diseases and to allow counseling and appropriate screening of relatives for a RET mutation. Even patients with MEN2B, which often have typical physical features, may not be properly recognized and be followed as a sporadic case. Based on this, all suspicious cases of multiple endocrine neoplasia should undergo a molecular genetic test.