The Effects Of Protein On Analysis Of Drug Concentration In Human Serum And Their Eliminate By Aqueous Two-Phase Systems

The most commonly reported method for the analysis drug concentration in humanserum involve two steps: the pre-treatment of samples and the detection of theconcentration of drugs. With the technology progress, the instruments can meet therequirements of the general determination. However, the key problem for accurate andeffective detection of drugs in serum is sample preparation. This paper aims at summaringthe existing preparation methods of drugs in serum. Studying the drug-human serumalbumin（HSA） interaction by fluorescence quenching method and different ethanolconcentration solution on protein denaturation by Rayleigh scattering, expounding theexisting state of drug in protein matrices sample. Further studying providing a theoreticalbasis for the extraction of drugs in protein matrix samples. Detailed study AqueousTwo-Phase Systems（ATPS） formation conditions of ethanol-K₂HPO₄-water, and thendetermination the alcohol content of alcoholic beverages with this theory. Based on the useof ethanol-K₂HPO₄-H2O ATPS approach and High Performance Liquid Chromatography（HPLC）, a novel method was developed using determination APDs and other drugs inhuman serum.The paper can be divided into five sections:Part one: In this paper, the pretreatment methods analysis drugs in human serumsamples and the use of ATPS extraction were reviewed, the background and significanceof this issue also were introduced.Part two: The effects of seven different inorganic salts on the phase separation inethanol-water solution have been studied. The results showed that K₂HPO₄was the bestsalt to form ATPS. Any volume fraction of ethanol-water system would be separated intoATPS when suitable amount of K₂HPO₄was added into the system, according to the phasediagrams of K₂HPO₄in ethanol-water system which was determined at25℃and normalatmospheric pressure. So the content of alcohol in alcoholic beverages can be determinedby measuring the volume of the up-phase of ATPS which was formed by adding salt in thebeverages. ADS-8resin and slightly chromate were added to eliminate the influence of anthocyanins and tannins on measures. The method been successfully used to determinethe content of alcohol in spirit, wine and brandy. The method was simple and quick. Theaccuracy of this method was evaluated by measuring the recovery from spiked samples.Overall recoveries were99.2%-101.6%with relative standard deviation values less than3.0%, which can meet the requirement for analysis in alcoholic beverages with thenominal content±1.0degrees.Part three: Using pure HSA as the model protein, the effects of protein on theextraction of antipsychotic drugs （APDs: diazepam, chlorpromazine hydrochloride,perphenazine） in Human Serum sample were studied. This paper detailedly investigatedthe interaction between APDs and HSA by fluorescence spectrometry, and differentethanol concentration solution on protein denaturation by Rayleigh scattering. The resultsshowed that APDs strongly bound with HSA. In the φ（ethanol）80%extracting solution, aslow but full protein denaturation takes place, which causes the unfolding of protein andthe dissociation of drugs. Then K₂HPO₄was added in extracting solution to form ATPS,and meanwhile the drugs was extracted into upper phase with high extraction efficiencies.After filtration with0.45μm micro-pore filter membrane, the upper phase was ready foranalysis by HPLC system. The detection limits were in the range of18.8³8.4ng·mL^-1,and the spiked recovery were94.2%⁹8.7%to determination APDs in human serum. Themethod is efficient, solvent-saving, environment-friendly, and accurate.Part four: Using pure HSA as the model protein, the effects of different ethanolconcentration solution on protein dissolve by fluorescence spectrometry, furtherdescription the influence of ethanol-H2O system to extraction drugs in human serum.Using ethanol-K₂HPO₄-H2O ATPS and HPLC extraction nifedipine, nitrendipine andnimodipine in human serum. The detection limits were in the range of10.4¹4.8ng·mL^-1,and the spiked recovery were86.2%⁹4.7%.Part five: Using ethanol-K₂HPO₄-H2O ATPS and HPLC extractionbendroflumethiazide, cyclopenthiazide and chlorprothixene in human serum.