Homemade Capillary Electrophoresis Coupled to a Mass Spectrometer

A system was developed in our laboratory to couple capillary electrophoresis with mass spectrometry. the capillary electrophoresis system was equipped with a high voltage supply, and a microcontroller with assembly language programming was developed for the computational control of the system. the MS system was a commercial Thermo Finningan LCQ ion trap mass spectrometer. the robustness of the coupled system was evaluated using standard protein samples (lysozyme, aprotinin, and bovine albumin) and tryptic digests of lysozyme. the system showed positive results in terms of robustness, allowing for the separation of digested proteins and the identification of 33% of the total amino acids in a protein (6 of the 18 expected peptides). the limit of detection was in the order of 1 picomole (signal-to-noise ratio), which was considered satisfactory for this system. the system shows high versatility in tandem coupling and combinations with other analytical procedures.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Dept Ciencias Exatas & Terra Diadema, São Paulo, BrazilUniv São Paulo, Inst Quim Sao Carlos, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Dept Ciencias Exatas & Terra Diadema, São Paulo, BrazilWeb of Scienc

Development of nanoinjector devices for electrospray ionization - tandem mass spectrometry (ESI-MSn)

In mass spectrometric (MS) systems with electrospray ionization (ESI), the sample can be analyzed coupled to separation systems (such as liquid chromatography or capillary electrophoresis) or simply by direct infusion. The greatest benefit of the type of injection is the possibility of continuous use of small amounts of samples over a long period of time. This extended analysis time allows a complete study of fragmentation by mass spectrometry, which is critical for structure elucidation of new compounds, or when using an ion trap mass analyzer. The injector filled with the sample is placed at the ESI source inlet creating an electric field suitable for the continuous formation of a spray (solvent and sample) and consequently, the gradual and even release of the sample. For the formation of the spray, is necessary that the injector end is metalized. The formation of a bilayer of titanium and gold provided an excellent attachment of the film, resulting in a nanoinjector for ionization/spray formation in the system for MS. The nanoinjectors showed high repeatability and stability over 100 min by continuous sampling with 10 µL of sample.Em espectrometria de massas (MS) no modo de ionização eletrospray (ESI), as amostras podem ser analisadas com um método prévio de separação (cromatografia líquida ou eletroforese capilar) ou por infusão direta da amostra. A injeção direta apresenta um grande benefício que é a redução do volume de amostra consumido e a possibilidade de amostragem contínua por um período estendido. Este maior tempo de amostragem possibilita a análise completa através de fragmentações sucessivas por espectrometria de massas, crítico quando se busca elucidação estrutural de novos compostos, ou quando se dispõe de um analisador de massas do tipo captura de íons. Neste trabalho, descrevemos uma metodologia de deposição de filme estável na extremidade cônica dos dispositivos de boro-silicato, visando o desenvolvimento de nanoinjetores estáticos. A formação de uma camada dupla de titânio e ouro proporcionou uma excelente fixação do filme, resultando em um nanoinjetor para amostragem por ionização/formação do aerossol no sistema de espectrometria de massas. O objetivo deste filme é manter o contato elétrico no sistema. Os nanoinjetores apresentaram repetibilidade e estabilidade elevadas por mais de 100 min de amostragem contínua com apenas 10 µL de amostra.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo (UNIFESP) Departamento de Ciências Exatas e da TerraUniversidade de São Paulo Instituto de Ciências BiomédicasUniversidade de São Paulo Instituto de Química de São CarlosUNIFESP, Depto. de Ciências Exatas e da TerraSciEL

Novo ENEM como Base para um Ensino Sustentável de Química no Brasil ( Área de Trabalho = CA )

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Potential diagnostic of Branched-Chain Ketoaciduria by HPLC-DAD

A system of high performance liquid chromatography (HPLC) was used for the development and validation of efficient method for quantitative determination of three aminoacids involved in the inherited metabolic disease Branched-Chain Ketoaciduria (BCK), also called maple syrup urine disease. The analytical conditions were selected in order to obtain baseline separation profiles of the amino acids known to be altered in blood plasma of BCK patients, namely L-valine, L-isoleucine, and L-leucine. Most accurate data were obtained using HPLC/diode detector. As the analytes do not have chromophore groups, they were pre-derivatized with o-phthaldialdehyde (OPA), yielding an unsaturated adduct, making thus possible the detection of amino acids. The validation was conducted according to National Health Surveillance Agency (ANVISA) and Guidance for Industry (Bioanalytical Method Validation) United States Food and Drug Administration (U.S. FDA). The results were satisfactory, with high sensitivity, good linearity, precision and accuracy, limit of detection and quantification, all within the established parameters for bioanalytical methods, showing its applicability and low cost compared to other existing techniques such as sequential mass spectrometry. For the three amino acids, L-valine, L-isoleucine and L-leucine, the detection limits (LOD) found were: 1.61, 1.84 and 1.88 mmol L- 1 and the quantification limits (LOQ) 4.37, 6.13 and 6.27 mmol L- 1, respectively.Neste trabalho, um sistema de cromatografia líquida de alta eficiência (HPLC) foi usado para desenvolver e validar um eficiente método para determinar quantitativamente aminoácidos envolvidos na desordem rara conhecida como cetonúria de aminoácidos de cadeia ramificada ou doença do xarope de bordo. As condições analíticas foram desenvolvidas para obter os perfis dos aminoácidos de L-valina, L-isoleucina e L-leucina, sabidamente alterados no plasma sanguíneo dos pacientes. Empregou-se HPLC provido de um detector com arranjo de diodo. Os analitos não possuem grupos cromóforos e, por isso, foram pré-derivatizados com o-ftalaldeído (OPA) para tornar possível sua detecção. A validação foi conduzida de acordo com as normas da Agência Nacional de Vigilância Sanitária (ANVISA) (RDC No. 27, de 17 de maio de 2012) e secção de validação de bioanalítica da United States Food and Drug Administration (U.S. FDA). Os resultados foram satisfatórios, apresentando alta sensibilidade, boa linearidade, precisão e exatidão, limite de detecção e quantificação, todos parâmetros estabelecidos para métodos bioanalíticos, demonstrando a aplicabilidade e baixo custo do método comparado com outras técnicas como espectrometria de massas. Para os três aminoácidos, L-valina, L-isoleucina e L-leucina, os limites de detecção encontrados foram: 1,61, 1,84 e 1,88 mmol L 1 e limites de quantificação 4,37, 6,13 e 6,27 mmol L 1, respectivamente.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Redoxoma (National Institute of Science and Technology of Redox Processes in Biomedicine)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Química e FarmacêuticasUniversidade de São Paulo Instituto de Química Departamento de Química FundamentalUNIFESP, Instituto de Ciências Ambientais, Química e Farmacêuticas2006/60245-3 e 2007/59039-2 e 2012/02514-9SciEL

Bioanalytical studies of porphyric disorders using HPLC with fluorescence detection

We describe here the development, validation, quantification and application of a method for determination of heme porphyrin precursors in the urine of porphyric patients. The isomers coproporphyrinogen I and III (COPRO I and III), uroporphyrinogen I (URO I), heptacarboxylporphyrinogen I (HEPTA I), pentacarboxylporphyrinogen (PENTA I), and hexacarboxylporphyrinogen I (HEXA I) were analyzed. These six urinary heme precursors were determined in urine samples collected from 24 patients by high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. The inter- and intra-day precision (coefficient of variation < 5%) and accuracy (95-99%) were evaluated. The limits of detection and of quantification of the porphyrins, expressed in nmol L-1, were as follows: URO I, 0.62 and 2.05; HEPTA I, 0.59 and 1.96; HEXA I, 0.54 and 1.81; PENTA I, 0.52 and 1.73; COPRO I, 2.03 and 6.77; and COPRO III, 0.43 and 1.44. The method described here satisfactorily results in an acceptable cost-benefit ratio, precision and speed for determining the concentrations of heme precursors in the urine of latent or symptomatic acute intermittent porphyria individuals or porphyria cutanea tarda carriers. Since it was analytically validated, this method may be used for accurate and reliable diagnostic reports to follow-up the onset of acute crisis in porphyria carriers to adopt preventive pharmacological treatment.Neste artigo, desenvolvemos, validamos e aplicamos um método para separação e quantificação de porfirinas precursoras do grupo heme na urina de portadores de porfirias. Os isômeros coproporfirinogenio I e III (COPRO I e III), uroporfirinogenio I (URO I), heptacarboxilporfirinogenio I (HEPTA I), pentacarboxilporfirinogenio I (PENTA I) e hexacarboxilporfirinogenio (HEXA I) foram determinados em amostras coletadas de 24 pacientes de porfiria aguda intermitente e de porfiria cutânea tarda. Utilizou-se cromatografia líquida de alta eficiência (HPLC) e detector de fluorescência. As concentrações de porfirinas foram determinadas com precisão inter e intra-dias (< 5%) e exatidão dentro da faixa 95-99%. Os limites de detecção e quantificação das porfirinas, expressos em nmol L-1, foram os seguintes: URO I, 0,62 e 2,05; HEPTA I, 0,59 e 1,96; HEXA I, 0,54 e 1,81; PENTA I, 0,52 e 1,73; COPRO I, 2,03 e 6,77; e COPRO III, 0,43 e 1,44. O método descrito aqui obedece a parâmetros analíticos satisfatórios, com excelente relação custo-benefício, e foi aplicado a amostras de urina de portadores assintomáticos e pacientes de porfirias. Este método foi validado analiticamente e mostrou potencial para diagnóstico de portadores de diferentes tipos de porfirias, imediatamente antes ou durante crises, e até mesmo para monitorar um tratamento farmacológico.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Redoxoma Redox Processes in BiomedicineFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Químicas e Farmacêuticas Departamento de Ciências Exatas e da TerraUniversidade de São Paulo Instituto de Química Departamento de Química FundamentalUNIFESP, Instituto de Ciências Ambientais, Químicas e Farmacêuticas Depto. de Ciências Exatas e da Terra2006/60245-3 e 2006/56530-4SciEL

Efeitos farmacocinéticos e clínicos de duas concentrações de bupivacaína no bloqueio do plexo braquial via axilar

Introduction: The risk of systemic bupivacaine toxicity is a persistent problem, which makes its pharmacokinetic study fundamental for regional anesthesia safety. There is little evidence of its influence on plasma peak at different concentrations. The present study compares two bupivacaine concentrations to establish how the concentration affects this drug plasma peak in axillary brachial plexus block. Postoperative latency and analgesia were also compared. Methods: 30 patients were randomized. In the 0.25% Group, 0.25% bupivacaine (10 mL) was injected per nerve. In the 0.5% Group, 0.5% bupivacaine (5 mL) was injected per nerve. Peripheral blood samples were collected during the first 2 hours after the blockade. For sample analyses, high performance liquid chromatography mass spectrometry was used. Results: Plasma peak occurred 45 minutes after the blockade, with no difference between groups at the assessed time-points. Plasma peak was 933.97 +/- 328.03 ng.mL(-1) (mean +/- SD) in 0.25% Group and 1022.79 +/- 253.81 ng.mL(-1) in 0.5% Group (p = 0.414). Latency was lower in 0.5% Group than in 0.25% Group (10.67 +/- 3.71 x 17.33min +/- 5.30, respectively, p = 0.004). No patient had pain within the first 4 hours after the blockade. Conclusion: For axillary brachial plexus block, there was no difference in bupivacaine plasma peak despite the use of different concentrations with the same local anesthetic mass. The concentration inversely influenced latency. (C) 2017 Published by Elsevier Editora Ltda. on behalf of Sociedade Brasileira de Anestesiologia.Introdução: O risco de intoxicação sistêmica pelo uso da bupivacaína é um problema persistente e torna seu estudo farmacocinético fundamental para a segurança da anestesia regional. São escassas as evidências sobre a influência de diferentes concentrações no pico plasmático desse fármaco. O presente estudo compara duas concentrações de bupivacaína para estabelecer como a concentração afeta o pico plasmático desse fármaco no bloqueio do plexo braquial via axilar. Também se compararam latência e analgesia pós-operatória. Métodos: Foram randomizados 30 pacientes. No Grupo 0,25%, injetaram-se 10 mL de bupivacaína 0,25% por nervo. No Grupo 0,5%, injetaram-se 5 mL de bupivacaína 0,5% por nervo. Amostras de sangue periférico foram colhidas durante as duas primeiras horas após o bloqueio. Para análise das amostras, usou-se a cromatografia líquida de alta frequência acoplada ao espectrômetro de massas. Resultados: O pico plasmático ocorreu 45 minutos após o bloqueio, sem diferença entre os grupos nos tempos avaliados. O pico plasmático (média ± DP) foi 933,97 ± 328,03 ng.mL−1 no Grupo 0,25% e 1.022,79 ± 253,81 ng.mL−1 no Grupo 0,5% (p = 0,414). O Grupo 0,5% apresentou menor latência com relação ao Grupo 0,25% (10,67 ± 3,71 × 17,33 min ± 5,30; respectivamente; p = 0,004). Nenhum paciente apresentou dor nas primeiras quatro horas após o bloqueio. Conclusão: Para o bloqueio do plexo braquial via axilar, não foi detectada diferença no pico plasmático de bupivacaína apesar do uso de diferentes concentrações, com a mesma massa de anestésico local. A concentração influenciou inversamente a latência.Univ Fed Sao Paulo Unifesp, Disciplina Anestesiol Dor & Terapia Intens, Sao Paulo, SP, BrazilUniv Fed Sao Paulo Unifesp, Inst Ciencias Ambientais Quim & Farmaceut, Sao Paulo, SP, BrazilUniv Fed Sao Paulo Unifesp, Disciplina Anestesiol Dor & Terapia Intens, Sao Paulo, SP, BrazilUniv Fed Sao Paulo Unifesp, Inst Ciencias Ambientais Quim & Farmaceut, Sao Paulo, SP, BrazilWeb of Scienc

Dysregulation of glycerophospholipid metabolism during Behçet's disease contributes to a pro-inflammatory phenotype of circulating monocytes

Behcet's disease (BD) is a relapsing, multisystem and inflammatory condition characterized by systemic vasculitis of small and large vessels. Although the etiopathogenesis of BD remains unknown, immune-mediated mechanisms play a major role in the development of the disease. BD patients present leukocyte infiltration in the mucocutaneous lesions as well as neutrophil hyperactivation. In contrast to neutrophils, whose involvement in the pathogenesis of BD has been extensively studied, the biology of monocytes during BD is less well known. In this study, we analyzed the phenotype and function of circulating monocytes of 38 BD patients from Hospital of Braga. In addition, we evaluated the impact of inflammatory and metabolomic plasma environment on monocyte biology. We observed a worsening of mitochondrial function, with lower mitochondrial mass and increased ROS production, on circulating monocytes of BD patients. Incubation of monocytes from healthy donors with the plasma of BD patients mimicked the observed phenotype, strongly suggesting the involvement of serum mediators. BD patients, regardless of their symptoms, had higher serum pro-inflammatory TNF-alpha and IP-10 levels and IL-1 beta/IL-1RA ratio. Untargeted metabolomic analysis identified a dysregulation of glycerophospholipid metabolism on BD patients, where a significant reduction of phospholipids was observed concomitantly with an increase of lysophospholipids and fatty acids. These observations converged to an enhanced phospholipase A2 (PLA(2)) activation. Indeed, inhibition of PLA(2) with dexamethasone or the downstream cyclooxygenase (COX) enzyme with ibuprofen was able to significantly revert the mitochondrial dysfunction observed on monocytes of BD patients. Our results show that the plasma inflammatory environment coupled with a dysregulation of glycerophospholipid metabolism in BD patients contribute to a dysfunction of circulating monocytes

Increased chemical acetylation of peptides and proteins in rats after daily ingestion of diacetyl analyzed by Nano-LC-MS/MS

Background. Acetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well. Methods. Wistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase colurnn and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOE-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data frommass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches. Results. A set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis. Conclusions. These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.Sao Paulo Research Foundation (FAPESP)Brazilian Innovation Agency (FINEP)Univ Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Diadema, SP, BrazilUniv Fed Sul & Sudeste Para, Inst Studies Hlth & Biol, Collect Hlth, Maraba, PA, BrazilFundacao Univ Fed Rondonia, Dept Chem, Porto Velho, RO, BrazilUniv Sao Paulo, Sao Carlos Inst Chem, Sao Carlos, SP, BrazilUniv Sao Paulo, Inst Chem, Dept Fundamental Chem, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Inst Environm Chem & Pharmaceut Sci, Diadema, SP, BrazilFAPESP: 2012/02514-9FAPESP: 2013/07763-0FAPESP: 2010/01404-0Web of Scienc

Is chemical exposure present in informal work associated with Sars-CoV-2 infection?

OBJECTIVE: To compare the incidence of Covid-19 symptoms between informal home-based workers and a control group and to assess the association of these cases with blood elements concentrations and other relevant risk factors for Sars-Cov-2 infection. METHODS: Welders chemically exposed to potentially toxic elements (PTEs) (n = 26) and control participants (n = 25) answered questionnaires on adherence to social distancing and signs and symptoms of the disease for five months during the Covid-19 pandemic. After follow-up, Covid-19 serology tests were performed on a subsample of 12 chemically exposed workers and 20 control participants. Before the pandemic, PTE concentrations in blood (As, Mn, Ni, Cd, Hg, Sb, Sn, Cu, Zn, and Pb) were measured by ICP-MS. RESULTS: The chemically exposed group had higher lead and cadmium levels in blood (p &lt; 0.01). The control group presented lower adherence to social distancing (p = 0.016). Although not significant, welders had a 74% greater chance of having at least one Covid-19 symptom compared with control participants, but their adherence to social distancing decreased this chance by 20%. The use of taxis for transportation was a risk factor significantly associated with Covid-19 symptoms. CONCLUSION: The lower adherence to social distancing among the control group greatly influences the development of Covid-19. The literature lacks data linking exposure to PTEs and Sars-Cov-2 infection and/or severity. In this study, despite chemical exposure, working from home may have protected welders against Covid-19, considering that they maintained greater social distancing than control participants