Desenvolvimento de metodologias analíticas para determinação de espécies de interesse clínico e biológico

Abstract

The development of new analytical methodologies for the determination of species of clinical and biological interest are constant demands of the Society. Therefore, the first work describes a colorimetric method for the determination of thimerosal (TM) in vaccines using paper-based analytical devices (PADs) as an analytical platform. TM is an organic mercury compound that is quantitatively converted to Hg(II) after oxidation by KMnO4 in an acid medium, thus Hg(II) reacts with dithizone to generate a colored complex. The proposed method presented a detection limit of 0,5 mg L-1 and a linear range of 1,7 - 11 mg L-1 with a relative standard deviation (RSD) < 3.6%, being selective for TM quantification in the presence of species commonly found in vaccines as Al(III), Na+ , K+ , Cl- , sulfate and phosphate salts. The method was applied to determine TM in different vaccines, with 89 to 112% recovery. The results obtained were statistically in agreement with the reference procedure based on microwave acid digestion and determination of total mercury by cold vapor atomic fluorescence spectroscopy (CV AFS). The proposed method to determine TM as total inorganic mercury was simple, fast, low cost, accurate and environmentally friendly (green); for providing low waste generation, in addition to being amenable to application in the quality control of vaccines. In the second work, a simple, fast and precise method was developed for the determination of total proteins, as a function of the concentration of human serum albumin (HSA) in biological samples using a fluorescent probe derived from heptamethine cyanine (SHP) based off-on mechanism with near infrared (NIR) emission. The proposed method was based on the absence of free probe fluorescence (SHP), however, the complex formed with the protein (SHP-HSA) shows high emission in NIR (λex = 580 nm / λem = 674 nm) under optimized conditions (pH 8.0 in 10 mM PIPES buffer). The stoichiometry of the HSA-SHP complex was defined as 1:1 applying Job's method. The linear range was 0,05 to 6,0 µM (equivalent to 3,4 - 408 mg L-1 ) with a detection limit of 0,014 µM (0,95 mg L-1 ) and RSD < 3.6%, being selective for quantification of HSA in the presence of species commonly found in biological samples such as metal ions, fatty acids, glucose and amino acids. Finally, the developed methodology was applied to determine SAH in plasma and urine samples, with recoveries from 91 to 118%, with the obtained results statistically concordant (α = 0,05) with the Bradford method (reference method). Thus, the proposed method for the determination of total proteins as a function of SAH proved to be versatile and amenable to application in the determination of total proteins for clinical purposes.