Desenvolvimento e validação de método analítico para determinação de WE010, WE014 e WE017

The synthetic guanylhydrazones WE010 (3,5-di-tert-butil-4-hidroxibenzaldehyde-guanylhydrazone), WE014 (4-bifenilcarboxialdehydeguanylhydrazone) and WE017 (3,4-diclorobenzaldehydeguanylhydrazone) showed high cytotoxic activity in terms of percentage inhibition of cancer cells growth. However, further progress in the development of these drug candidates requires precise and convenient methods for their qualitative and quantitative analyses. The aim of this study was to develop and validate High Performance Liquid Chromatography with diode-array detection (HPLC-DAD) and Ultra Fast Liquid Chromatography with diode-array detection (UFLC-DAD) methods suitable for as simultaneous as isolated determination of studied guanylhydrazones, based on the optimization of chromatographic parameters and obtaining reduced detection times. The chromatographic analyses of analytes by HPLC were performed on C18 ACE analytical column (150 mm x 4.6 mm), with a particle size of 5.0 μm. Among all the conditions assayed, the best results of separation were obtained with a mixture of methanol:water (60:40, v/v) as the mobile phase at a flow rate 1.5mL/min and pH of 3.5 adjusted at acetic acid. The UFLC method was developed by experimetal desing techniques in order to find optimal chromatographic analytical conditions, which were achieved on XR-ODS analytical column (50 mm x 3.0 mm), with a particle size of 2,2 μm, maintained at 25 ºC. The mobile phase was consisted of methanol:water (65:35, v/v) with 0.1% triethylamine (TEA) and pH of 3.5 adjusted at acetic acid, at a flow rate 0.5 mL/min. The procedure were validated following evaluating parameters such as specificity, linearity, limits of detection (LD) and quantification (LQ), precision, accuracy and robustness, giving results within the acceptable range. Although the UFLC method shows better sensitivity (lower values of LD and LQ), robustness (lower rates of relative standard deviation) and minimize spending time and solvent, both developed methods were adequately applied to the analysis of guanylhydrazones molecules, may be used in routine of quality control laboratories. Keywords: guanylhydrazones, HPLC/DAD, UFLC/DAD, validation of analitical method

Effect of supplementation of diets for quails with vitamins A, D and E on performance of the birds and quality and enrichment of eggs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

High concentrations of the melatonin metabolite, N-1-acetyl-N (2)-formyl-5-methoxykynuramine, in cerebrospinal fluid of patients with meningitis: a possible immunomodulatory mechanism

We evaluated the presence of the melatonin metabolite N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK), in cerebrospinal fluid (CSF) of patients with viral meningitis (n = 20) and control samples (n = 8) and correlate AFMK levels with inflammatory markers such as cellularity, protein, tumor necrosis factor (TNF)-alpha, interleukin (IL)-8 and IL-1 beta levels. A portion of the CSF was extracted with dichloromethane (1:5) and analyzed by high-performance liquid chromatography (HPLC) under standardized conditions for AFMK. AFMK was detected in 16 of 20 CSF samples of patients with viral meningitis; the concentration of AFMK was found to be above the quantification limit (50 nmol/L) in six of these samples. AFMK was not detected in any of the eight control samples. The samples were classified into groups according to AFMK levels: undetectable (< 10 nmol/L, group I), detectable but below the quantification limit (< 50 nmol/L, group II), and quantified (> 50 nmol/L, group III). Group II presented the highest levels of proteins and IL-8, whereas group III showed the lowest levels of the inflammatory parameters. This study supports our hypothesis that inflammation favors the formation of AFMK and that this compound has immunomodulatory activity in vivo.

The identification of beta-thalassemia mutants in Brazilians with high Hb F levels

As hemoglobinopatias são um grupo de afecções genéticas que representam problema de saúde pública em muitos países em que sua incidência é alta, com significativa morbidade. Objetivamos identificar defeitos moleculares que pudessem explicar o perfil laboratorial obtido por eletroforese e HPLC com Hb F elevada, em um grupo de indivíduos adultos sem sinais ou sintomas de anemia. Encontramos cinco diferentes mutações que originam beta talassemia por PCR-ASO: três casos com CD 6 (-A), um CD 39, um IVS 1-5, um -87 todas de origem mediterrânea, e um IVS II-654 de origem asiática. As mutações CD 6 (-A), -87 e IVS II-654 foram descritas pela primeira vez na população brasileira.

Determination of 2,4-dichlorophenoxyacetic acid and its major transformation product in soil samples by liquid chromatographic analysis

The 2,4-dichlorophenoxyacetic acid (2,4-D) is one of the most applied herbicides around the world to control broad leave herbs in many crops: In this study, a method was developed for simultaneous extraction and determination of 2,4D and its major transformation product, i.e., the 2,4-dichlorophenol (2,4-DCP), in soil samples. The herbicide and its degradation product were extracted twice from soil samples, after acidification, by dichloromethane on ultrasound system for 1 h. Both extracts were combined and filtrated in qualitative filter paper and Celitee. The total extract was concentrated in rotatory evaporator, dried under N-2 and finally dissolved in 1 ml of methanol. High Performance Liquid Chromatography with UV detection at 230 nm was used for analysis. Recoveries were obtained from soil samples fortified at 0.1, 1.0, 2.0, 3.0 and 4.0 mg kg(-1) levels and the results varied from 85 to 111% (for 2,4-D) and from 95 to 98% (for 2,4-DCP). For both compounds, the limits of quantification were 0.1 mg kg(-1), which were the loss level at which the accuracy and the precision were studied. Nevertheless, the limits of detection, calculated by considering the blank standard deviation and the minimum concentration level, were 0.03 and 0.02 mg kg for 2,4-D and 2,4-DCP, respectively. This proposed method was applied to soil samples of eucalyptus crops, which was previously treated with the herbicide. Despite that, neither 2,4-D nor its degradation product were detected 30 days after the herbicide application. (C) 2003 Elsevier B.V. B.V. All rights reserved.

A comparison of mycolic acid analysis for nontuberculous mycobacteria identification by thin-layer chromatography and molecular methods

The development of fast, inexpensive, and reliable tests to identify nontuberculous mycobacteria (NTM) is needed. Studies have indicated that the conventional identification procedures, including biochemical assays, are imprecise. This study evaluated a proposed alternative identification method in which 83 NTM isolates, previously identified by conventional biochemical testing and in-house M. avium IS1245-PCR amplification, were submitted to the following tests: thin-layer chromatography (TLC) of mycolic acids and PCR-restriction enzyme analysis of hsp65 (PRA). High-performance liquid chromatography (HPLC) analysis of mycolic acids and Southern blot analysis for M. avium IS1245 were performed on the strains that evidenced discrepancies on either of the above tests. Sixty-eight out of 83 (82%) isolates were concordantly identified by the presence of IS1245 and PRA and by TLC mycolic acid analysis. Discrepant results were found between the phenotypic and molecular tests in 12/83 (14.4%) isolates. Most of these strains were isolated from non-sterile body sites and were most probably colonizing in the host tissue. While TLC patterns suggested the presence of polymycobacterial infection in 3/83 (3.6%) cultures, this was the case in only one HPLC-tested culture and in none of those tested by PRA. The results of this study indicated that, as a phenotypic identification procedure, TLC mycolic acid determination could be considered a relatively simple and cost-effective method for routine screening of NTM isolates in mycobacteriology laboratory practice with a potential for use in developing countries. Further positive evidence was that this method demonstrated general agreement on MAC and M. simiae identification, including in the mixed cultures that predominated in the isolates of the disseminated infections in the AIDS patients under study. In view of the fact that the same treatment regimen is recommended for infections caused by these two species, TLC mycolic acid analysis may be a useful identification tool wherever molecular methods are unaffordable.

Recent applications of analytical techniques for quantitative pharmaceutical analysis: A review

The intension of this paper was to review and discuss some of the current quantitative analytical procedures which are used for quality control of pharmaceutical products. The selected papers were organized according to the analytical technique employed. Several techniques like ultraviolet/visible spectrophotometry, fluorimetry, titrimetry, electroanalytical techniques, chromatographic methods (thin-layer chromatography, gas chromatography and high-performance liquid chromatography), capillary electrophoresis and vibrational spectroscopies are the main techniques that have been used for the quantitative analysis of pharmaceutical compounds. In conclusion, although simple techniques such as UV/VIS spectrophotometry and TLC are still extensively employed, HPLC is the most popular instrumental technique used for the analysis of pharmaceuticals. Besides, a review of recent works in the area of pharmaceutical analysis showed a trend in the application of techniques increasingly rapid such as ultra performance liquid chromatography and the use of sensitive and specific detectors as mass spectrometers.

Nasal administration of liquid crystal precursor mucoadhesive vehicle as an alternative antiretroviral therapy

The purpose of this study was to develop a mucoadhesive stimuli-sensitive drug delivery system for nasal administration of zidovudine (AZT). The system was prepared by formulating a low viscosity precursor of a liquid crystal phase, taking advantage of its lyotropic phase behavior. Flow rheology measurements showed that the formulation composed of PPG-5-CETETH-20, oleic acid and water (55, 30, 15% w/w), denominated P, has Newtonian flow behavior. Polarized light microscopy (PLM) revealed that formulation P is isotropic, whereas its 1:1 (w/w) dilution with artificial nasal mucus (ANM) changed the system to an anisotropic lamellar phase (PD). Oscillatory frequency sweep analysis showed that PD has a high storage modulus (G′) at nasal temperatures. Measurement of the mucoadhesive force against excised porcine nasal mucosa or a mucin disk proved that the transition to the lamellar phase tripled the work of mucoadhesion. Ex vivo permeation studies across porcine nasal mucosa exhibited an 18-fold rise in the permeability of AZT from the formulation. The Weibull mathematical model suggested that the AZT is released by Fickian diffusion mechanisms. Hence, the physicochemical characterization, combined with ex vivo studies, revealed that the PPG-5-CETETH-20, oleic acid, and water formulation could form a mucoadhesive matrix in contact with nasal mucus that promoted nasal absorption of the AZT. For an in vivo assessment, the plasma concentrations of AZT in rats were determined by HPLC method following intravenous and intranasal administration of AZT-loaded P formulation (PA) and AZT solution, respectively, at a dose of 8 mg/kg. The intranasal administration of PA resulted in a fast absorption process (Tmax = 6.7 min). Therefore, a liquid crystal precursor formulation administered by the nasal route might represent a promising novel tool for the systemic delivery of AZT and other antiretroviral drugs. In the present study, the uptake of AZT absorption in the nasal mucosa was demonstrated, providing new foundations for clinical trials in patients with AIDS. © 2012 Elsevier B.V. All rights reserved.

Ocorrência de isoflavonas de soja no ambiente e correlação com atividades estrogênica : estudo de caso da região de Dourados (MS)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A trade off between separation, detection and sustainability in liquid chromatographic fingerprinting

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo químico e atividade mutagênica e antirradicalar de caesalpinia ferrea

Pós-graduação em Química - IQ

Validação e aplicação de método para análise de pesticidas em água para consumo humano de dourados (ms) por CLAE/UV e CG/DTE

Indiscriminate and inappropriate use of pesticides in agriculture has been pointed out for increasing health problems and environmental damage. Considering that water resources are the principal destiny of those compounds after application, the present study presents optimization and validation of two simple and effi cient analytical methods for pesticides quantifi cation in both surface and groundwater. Were selected the pesticides more commonly used at Dourados (MS - Brazil), region with intense agricultural activity. Pesticides were preconcentrated by solid-phase extraction using C18 (500 mg) cartridges and then divided in two groups for elution and quantifi cation: 2.4-D and 2.4-DCP were eluted with methanol and quantifi ed by high performance liquid chromatography with ultra-violet detector (HPLC-UV) while atrazine, DIA, DEA, trifl uralin and methyl parathion were eluted with ethylacetate (1:1, v/v) and quantifi ed by gas chromatography with thermionic specifi c detector (GC-TSD). The methods showed satisfactory accuracy (76-107%) and precision (<12%) for the substances analyzed at the fortifi ed levels selected for the study, except for DIA (<51%). Study of pesticide stability also presented good results: C18 cartridges could be stored for at least for 21 days at -20ºC with no signs of the compounds degradability. Both methods limits of quantifi cation of the pesticides (0.22 - 0.48 μg L-1) are in accordance to the levels currently established by the Brazilian national legislation for pesticides in water. Although only the pesticide 2.4-D has been detected in two distinct collection points in the study period of time, this work warns for the requirement of systematical analysis of pesticides presence in water destined to human consume, principally in areas of intense agriculture activity. Such monitoring can provide subsidies for public environmental policies.

Cromatografia Verde

Sustainability is the ability of human beings interact with the world, preserving the environment to not compromise the natural resources of future generations. This is the main goal of Green Chemistry, which is based on twelve basic principles. One of its branches, the Green Analytical Chemistry, apply these principles in their techniques, such as Green Chromatography. The aim of this study was to do a brief literature review on some chromatographic techniques (Gas Chromatography, High Performance Liquid Chromatography, High Temperature Liquid Chromatography, Ultra Pressure Liquid Chromatograph, Micro Liquid Chromatography and Super Critical Chromatography) and measure their impact on the environment

Acetone as a greener alternative to acetonitrile in liquid chromatographic fingerprinting

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determinação do teor dos ingredientes ativos imidacloprida e clorpirifós em produtos técnicos formulados por CLUE-DAD

The use of pesticides to manage pest problems in agriculture has become a common practice around the world. Pesticides consist in chemical substances or biological agents that act preventing, destroying, repelling or mitigating the damage caused by pests in agriculture. In order to a successful treatment, the correct dosage of pesticide has to be applied for each kind of culture. In this context, this project aims to quantify the levels of the active ingredients Imidacloprid and Chlorpyrifos present in formulated technical products and compare with the reported in the ir commercial leaflets. The analyzes were performed by Ultra Performance Liquid Chromatography coupled to a photodiode array detector (CLUE-DAD). The identification of Imidacloprid and Chlorpyrifos in the samples was realized by comparison of the time of retention and UV spectre of those substances with the commercials patterns, resulting in a excellent method specificity of the method. In the scope of determination of active ingredients in formulated products, the method developed showed good sensitivity in the applied conditions, with suitables limits of detection (4,2.10-² mg L-¹ for Imidacloprid and 1,1.10-¹ mg L-¹ to Chlorpyrifos) and quantitation (1,4.10-¹ mg L-¹ and Imidacloprid 3,6.10-¹ mg L-¹ to Chlorpyrifos) for the concentrations found in matrices analyzed. Furthermore, good results were obtained to the method accuracy with coeficiente of variance values in the range of 0,6 to 0,7% for repeatability (intra-day) and 0,4 to 0,58% for intermediate precision (inter- day). The accuracy of the method was determined by recovery tests, the values were comprised in the range of 98 102%. According to the recommendations of the Associação Brasileira de Normas Técnicas, these values show good accuracy of the method. The concentrations obtained in the quantification of the active ingredients in formulated products showed values within permited by ABNT, 60.1% (w/v) ± 0.6%...