On-line multidimensional separation systems for peptide analysis

Today, there is an increasing interest in selective and sensitive analysis of proteins and peptides with a relatively high speed. The first chapter of this thesis describes several strategies for the on-line multidimensional analysis of peptides and proteins in biological samples. This overview of the existing approaches to couple on-line different separation techniques for the determination of proteins and peptides includes the more conventional liquid chromatography (LC)-LC coupling, as well as the on-line LC-capillary electrophoresis (CE) coupled systems and the strategies for on-line CE-CE coupling. Special attention is paid to the interface between the dimensions as well as the design of the coupled systems. An overview of the possibilities of on- or in-line preconcentration procedures in combination with a CE separation, focused on the determination of peptides and proteins, is also included in the thesis (chapter 2). The methods are categorized in electrophoresis-based and chromatography-based preconcentration. The objective of this thesis is the development of novel on-line multidimensional systems for the separation and quantitation of peptides in biological samples. Two on-line two-dimensional (2D) systems have been developed: a size exclusion chromatography (SEC)-reversed phase liquid chromatography (RPLC) system (chapter 3-4) and a SEC-capillary zone electrophoresis (CZE) system (chapter 5-6). In both cases the heart-cut principle is used to isolate in the SEC dimension the fraction, containing six enkephalin peptides as target compounds, from proteins and other interfering substances. This fraction is transferred to the second (RPLC or CZE) dimension and its components are separated. In the SEC-RPLC system a loop is used to transfer the enkephalin-containing fraction, while the SEC-CZE system is based on a new type of interface consisting of trapping of the enkephalins on a micro column and on-line electrokinetic injection of a part of the eluate from this column. Determination of model enkephalins, developed with both systems show satisfactory linearity and intraday precision while the total analysis time is less than 30 min. The concentration limit of detection (CLOD) of both systems, employing UV detection, is in the order of 2-3 μg/mL, which is comparable to other peptide assays based on separation and suitable for the determination and quantitation of a number of peptide drugs with plasma concentrations in the low μg/mL range. Both methods allow the injection of CSF samples without pretreatment. However, due to the limited sensitivity of the methods the concentrations of endogenous enkephalins in plasma and CSF cannot be determined, since these concentrations are at least three orders of magnitude lower than the presented CLOD values. It is therefore necessary to improve the sensitivity of the systems. In the general conclusions of this thesis preliminary experiments with more sensitive detection methods, such as fluorescence and MS detection are described in order to illustrate the possibilities and difficulties of the coupling with these detection modes. Some recommendations are given for the achievement of a higher sensitivity