Evaluation of the relative efficacy of various techniques for deproteinizing plasma samples prior to high-performance liquid chromatographic analysis.

The methods used for treating biological samples prior to their introduction into a high-performance liquid chromatographic (HPLC) system generally fall into one of two categories extraction or direct injection. In the extraction method the compound of interest is removed from the biological matrix (plasma, serum, urine, etc.) using suitable solvent and pH conditions, which selectively extract the desired components and leave behind unwanted materials. The solvent is then removed by gentle evaporation and the dried residue reconstituted in a small volume of the elution solvent (or one quite similar to it) for injection on to the HPLC column. The direct-injection technique is by far the simplest and most rapid of the two methods_ In this procedure the biological sample may be injected directly on to the top of the HPLC column cl] _ However, a number of reports have indicated that this results in a rapid increase in back-pressure -and a deterioration of column performance [Z-5], presumably due to the precipitation of plasma proteins as a result of their contact with the organic solvents and buffer salts commonly utilized in mobile phases [4]_ To alleviate this problem, a number of sample preparation techniques have been described for removing proteins prior to. injection of the sample. These include the use of precolumns [S] , ultrafiltration devices [7, 81, and various protein precipitants such as organic solvents [9-111 and ionic salts [X2-14] _ Only one report dealing -specifically with sample preparation procedures for the direct-injection HPLC technique has appeared to date [15] _ In that study, -six. different methods of deproteinizing plasma were evaluated using the -biuret assay to assess their efficiency. In the present report we describe a number of other potentially useful methods of protein removal,. using the much moresensitive Lowry [ 161 method of protein determination to evaluate :