Methodological features of the spectrophotometric determination of proteins in biological fluids using reactions with brompyrogallol red

Abstract

Determination of proteins in biological fluids is rather important for diagnostics in current clinical practice. The results of total protein determination depend on the amino-acid composition of the proteins present in the biological fluid. We discuss some aspects of the spectrophotometric determination of proteins in biological fluids, in particular, the methodological features of the technique based on the reaction of proteins with brompyrogallol red (BPGR). The most important advantage of BPGR in the determination of proteins in biological fluids is rather high and equal sensitivity of the dye to the proteins of albumin and globulin fractions, thus minimizing the errors attributed to the mismatch of the protein composition of the analyzed samples and calibration solutions used. The goal of the work is to study the impact of conditions and shelf life of the BPGR solution on the analytical properties of the solution in the spectrophotometric determination of proteins in biological fluids. Stability of the optical and analytical properties of BPGR solutions are studied using Fisher and Student criteria under conditions of different storage temperatures and nature of the stabilizer (ethanol or sodium benzoate) in the reagent solutions. Verification of the correctness of the total protein determination by the proposed method was carried out in spike tests. The introduced additives of standard solutions are prepared from the «Total protein» or «Albumin» calibrators. The developed method of the spectrophotometric determination of the mass concentration of proteins in the urine by the reaction with bromopyrogallol red was tested on real objects, metrologically certified and listed in the Federal register of certified measurement techniques. Analytical and metrological studies have shown that the developed method of protein determination with a reagent based on BPGR provides equal and high sensitivity of determination of albumin and globulin protein fractions in human biological fluids. To increase the shelf life of the reagent solution and preserve the analytical properties of the solution, we recommend to use ethanol as a stabilizer.