Residual protein content in the blood plasma biosamples of laboratory animals (rabbits) after their preparation for HPLC-UV analysis

Relevance. The main limitation of the sensitivity of HPLC-UV techniques widely used for the analysis of drugs in biological samples (blood plasma, tissues and organs) is due to the significant background influence of biologic matrices. Precipitation of proteins is the simplest, most versatile and reasonably efficient way to prepare biosamples for HPLC analysis. Information on the effectiveness of the protein deposition methods used and the level of their residual content in the analyzed biosamples is limited in the available literature. The aim: to evaluate the residual protein content in the blood plasma biosamples of laboratory animals (rabbits) after samples preparation for HPLC-UV analysis. Material and methods. The residual protein content in the rabbits blood plasma biosamples was evaluated by spectroscopic methods after samples treatment for HPLC-UV analysis, including precipitation with acidic solutions, acetonitrile and methanol. Results. The Bredford method has been shown to produce lower level results compared to the direct spectrophotometry method. Using different precipitation agents, their ratios and two centrifugation modes, the residual protein content determined by the Bradford method was about 0.02-0.4 mg/ml, indicating almost complete precipitation of the proteins (more than 99.5%) and confirming the correctness of using the precipitation sample preparation for further HPLC-UV analysis of the samples. Most preferred in terms of minimum residual protein content is the use of acetonitrile as a precipitation agent. The highest level of protein was found in the samples after treatment with 15% chloric acid, i.e. the use of this precipitation agent is least desirable for further HPLC analysis. Conclusions. Precipitation is effective way for sample protein removing; established features of various precipitation agent application may be used for development of bioanalytical methods, based on HPLC-analysis.

plasma, rat, sample treatment, protein, residue quantification