Preparo de padrões analíticos, estudo de estabilidade e parâmetros de validação para ensaio de carotenóides por cromatografia líquida

Abstract

Vitamin A is essential to the human beings, being ingested through animal sources as meat and eggs, or gotten for the conversion of pro vitamin carotenoids found mainly in fruits and vegetables. The vitamin A deficiency is the major nutritional problem that affects underdeveloped populations. As a consequence of this deficiency, xerophthalmia, blindness and premature death can occur, mainly in children. Vitamin A is also important for growth maintenance, for reproductive efficiency, maintenance of the epithelial tissues and keratinization prevention, beyond presenting an important role into the immunological response. Nowadays, the deficient intake of vitamin A is the major nutritional problem found into developing countries populations or poor population of the developed countries. Around of 4.4 million of children, within preschool age, are presenting vision damages due to the deficiency of this vitamin, every year about 250-500 thousand children become blind. Carotenoids are the most important source of retinol for populations in which the consumption of this vitamin is low. For this reason, quantitative and qualitative analysis of carotenoids in foods is very important. In this work, a specific chromatographic methodology was developed to separate the carotenoids and their isomers. Chromatographic separation of the main food carotenoids was optimized and validated. The chromatographic standards were isolated with appropriated amount and purity, and their stability was evaluated to determine the better conditions of storage, as well as shelf life. The isolation of all chromatographic standards was carried out with open column, quantification was done using spectrophotometry and purity evaluation was measured by HPLC. Many matrices have been evaluated as a source of standards, some new sources were found and showed better characteristics, as purity higher than 90%, if compared with those, frequently, used for this purpose. The study of stability showed that mixtures of carotenoids used as external standard for calibration can be storage more than 370 days. Validation study showed that this methodology of analysis has good selectivity, sensitivity and linearity in a concentration range from 0.5 to 8.0 μg/mL for each carotenoid.