Desenvolvimento e validação de método analítico para determinação de teor de ácido gálico e catequina no fitoterápico sanativo® por cromatografia líquida de alta eficiência

The herbal medicine Sanativo® is produced by the Pernambucano Laboratory since 1888 with indications of healing and hemostasis. It is composed of a fluid extract about Piptadenia colubrina, Schinus terebinthifolius, Cereus peruvianus and Physalis angulata. Among the plants in their composition, S. terebinthifolius and P. colubrina have in common phenolic compounds which are assigned most of its pharmacological effects. The tannins, gallic acid and catechin were selected as markers for quality control. The aim of this study was the development and validation of analytical method by HPLC/UV/DAD for the separation and simultaneous quantification of gallic acid (GAC) and catechin (CTQ) in Sanativo®. The chromatographic system was to stationary phase, C-18 RP column, 4,6 x 150 mm (5 mm) under a temperature of 35 ° C, detection at 270 and 210 nm. The mobile phase consisted of 0.05% trifluoroacetic acid and methanol in the proportions 88:12 (v/v), a flow rate of 1 ml/min. The analytical method presented a retention factor of 0.30 and 1.36, tail factor of 1.8 and 1.63 for gallic acid and catechin, respectively, resolution of 18.2, and theoretical plates above 2000. The method validation parameters met the requirements of Resolution n º 899 of May 29, 2003, ANVISA. The correlation coefficient of linear regression analysis for GAC and CTQ from the standard solution was 0.9958 and 0.9973 and when performed from the Sanativo® 0.9973 and 0.9936, the matrix does not interfere in the range 70 to 110 %. The limits of detection and quantification for GAC and CQT were 3.25 and 0.863, and 9.57 and 2.55 mg/mL, respectively. The markers, GAC and CQT, showed repetibility (coefficient of variation of 0.94 % and 2.36 %) and satisfactory recovery (100.02 ± 1.11 % and 101.32 ± 1.36 %). The method has been characterized selective and robust quantification of GAC and CTQ in the Sanativo® and was considered validated

Avaliação de um método de cromatografia em fase gasosa head space e estudo da estabilidade do etanol em amostras de sangue

The ethanol is the most overused psychoactive drug over the world; this fact makes it one of the main substances required in toxicological exams nowadays. The development of an analytical method, adaptation or implementation of a method known, involves a process of validation that estimates its efficiency in the laboratory routine and credibility of the method. The stability is defined as the ability of the sample of material to keep the initial value of a quantitative measure for a defined period within specific limits when stored under defined conditions. This study aimed to evaluate the method of Gas chromatography and study the stability of ethanol in blood samples, considering the variables time and temperature of storage, and the presence of preservative and, with that check if the conditions of conservation and storage used in this study maintain the quality of the sample and preserve the originally amount of analyte present. Blood samples were collected from 10 volunteers to evaluate the method and to study the stability of ethanol. For the evaluation of the method, part of the samples was added to known concentrations of ethanol. In the study of stability, the other side of the pool of blood was placed in two containers: one containing the preservative sodium fluoride 1% and the anticoagulant heparin and the other only heparin, was added ethanol at a concentration of 0.6 g/L, fractionated in two bottles, one being stored at 4ºC (refrigerator) and another at -20ºC (freezer), the tests were performed on the same day (time zero) and after 1, 3, 7, 14, 30 and 60 days of storage. The assessment found the difference in results during storage in relation to time zero. It used the technique of headspace associated with gas chromatography with the FID and capillary column with stationary phase of polyethylene. The best analysis of chromatographic conditions were: temperature of 50ºC (column), 150ºC (jet) and 250ºC (detector), with retention time for ethanol from 9.107 ± 0.026 and the tercbutanol (internal standard) of 8.170 ± 0.081 minutes, the ethanol being separated properly from acetaldehyde, acetone, methanol and 2-propanol, which are potential interfering in the determination of ethanol. The technique showed linearity in the concentration range of 0.01 and 3.2 g/L (0.8051 x + y = 0.6196; r2 = 0.999). The calibration curve showed the following equation of the line: y = x 0.7542 + 0.6545, with a linear correlation coefficient equal to 0.996. The average recovery was 100.2%, the coefficients of variation of accuracy and inter intra test showed values of up to 7.3%, the limit of detection and quantification was 0.01 g/L and showed coefficient of variation within the allowed. The analytical method evaluated in this study proved to be fast, efficient and practical, given the objective of this work satisfactorily. The study of stability has less than 20% difference in the response obtained under the conditions of storage and stipulated period, compared with the response obtained at time zero and at the significance level of 5%, no statistical difference in the concentration of ethanol was observed between analysis. The results reinforce the reliability of the method of gas chromatography and blood samples in search of ethanol, either in the toxicological, forensic, social or clinic

Influência do estresse nutricional programado na composição da microalga isochrysis galbana

Global warming due to Greenhouse Gases (GHG) emissions, especially CO2, has been identified as one of the major problems of the twenty-first century, considering the consequences that could represent to planet. Currently, biological processes have been mentioned as a possible solution, especially CO2 biofixation due to association microalgae growth. This strategy has been emphasized as in addition to CO2 mitigation, occurs the production of biomass rich in compounds of high added value. The Microalgae show high photosynthetic capacity and growth rate higher than the superior plants, doubling its biomass in one day. Its culture does not show seasons, they grow in salt water and do not require irrigation, herbicides or pesticides. The lipid content of these microorganisms, depending on the species, may range from 10 to 70% of its dry weight, reaching 90% under certain culture conditions. Studies indicate that the most effective method to promote increased production of lipids in microalgae is to induce stress by limiting nitrogen content in the culture medium. These evidences justify research continuing the production of biofuels from microalgae. In this paper, it was studied the strategy of increasing the production of lipids in microalgae I. galbana with programmed nutritional stress, due to nitrogen limitation. The physiological responses of microalgae, grown in f / 2 with different concentrations of nitrogen (N: P 15,0-control, N: 5,0 P and N: P 2,5) were monitored. During exponential phase, results showed invariability in the studied conditions. However the cultures subjected to stress in stationary phase, showed lower biomass yields. There was an increase of 32,5% in carbohydrate content and 87.68% in lipids content at N: P ratio of 5,0 and an average decrease of 65% in protein content at N: P ratios of 5, 0 and 2.5. There were no significant variations in ash content, independently of cultivation and growth phase. Despite the limitation of biomass production in cultures with N: P smaller ratios, the increase of lipid accumulation highest lipids yields were observed as compared to the control culture. Given the increased concentration of lipids associated to stress, this study suggests the use of microalgae Isochrysis galbana as an alternative raw material for biofuel production

Otimização evalidação de métodos analíticos para a determinação simultânea de tuberculostáticos (4-FDC) por CLAE/DAD e CLUE/ DAD

Tuberculosis is a serious disease, but curable in practically 100% of new cases, since complied the principles of modern chemotherapy. Isoniazid (ISN), Rifampicin (RIF), Pyrazinamide (PYR) and Chloride Ethambutol (ETA) are considered first line drugs in the treatment of tuberculosis, by combining the highest level of efficiency with acceptable degree of toxicity. Concerning USP 33 - NF28 (2010) the chromatography analysis to 3 of 4 drugs (ISN, PYR and RIF) last in average 15 minutes and 10 minutes more to obtain the 4th drug (ETA) using a column and mobile phase mixture different, becoming its industrial application unfavorable. Thus, many studies have being carried out to minimize this problem. An alternative would use the UFLC, which is based with the same principles of HPLC, however it uses stationary phases with particles smaller than 2 μm. Therefore, this study goals to develop and validate new analytical methods to determine simultaneously the drugs by HPLC/DAD and UFLC/DAD. For this, a analytical screening was carried out, which verified that is necessary a gradient of mobile phase system A (acetate buffer:methanol 94:6 v/v) and B (acetate buffer:acetonitrile 55:45 v/v). Furthermore, to the development and optimization of the method in HPLC and UFLC, with achievement of the values of system suitability into the criteria limits required for both techniques, the validations have began. Standard solutions and tablets test solutions were prepared and injected into HPLC and UFLC, containing 0.008 mg/mL ISN, 0.043 mg/mL PYR, 0.030 mg.mL-1 ETA and 0.016 mg/mL RIF. The validation of analytical methods for HPLC and UFLC was carried out with the determination of specificity/selectivity, analytical curve, linearity, precision, limits of detection and quantification, accuracy and robustness. The methods were adequate for determination of 4 drugs separately without interfered with the others. Precise, due to the fact of the methods demonstrated since with the days variation, besides the repeatability, the values were into the level required by the regular agency. Linear (R> 0,99), once the methods were capable to demonstrate results directly proportional to the concentration of the analyte sample, within of specified range. Accurate, once the methods were capable to present values of variation coefficient and recovery percentage into the required limits (98 to 102%). The methods showed LOD and LOQ very low showing the high sensitivity of the methods for the four drugs. The robustness of the methods were evaluate, facing the temperature and flow changes, where they showed robustness just with the preview conditions established of temperature and flow, abrupt changes may influence with the results of methods