Development of an on-line size exclusion chromatographic — reversed-phase liquid chromatographic two-dimensional system for the quantitative determination of peptides with concentration prior to reversed-phase liquid chromatographic separation Original Re

Complex samples, containing endogenous peptides require analytical methods with high sensitivity and selectivity to enable reliable analysis of these compounds in biological samples. In a number of cases, the selectivity of mono-dimensional separation systems is not sufficient. In order to improve the separation efficiency two- or multi-dimensional systems can be the solution. The present study aims at the development and validation of an on-line two-dimensional separation system, using size exclusion chromatography (SEC) and reversed-phase liquid chromatography (RP-LC), to quantitate structurally related peptides in the presence of large proteins such as albumin in contrast with the available coupled-column systems which are focussed on the qualitative determination of peptides. As model peptides a number of enkephalins have been chosen. Albumin was added to mimict a real biological sample. If the SEC dimension, the RP-LC dimension and the interface have been chosen properly, the entire peak, eluting from the SEC column and containing the enkephalins, can be trapped in a loop after complete separation from albumin. Subsequent separation of the enkephalins is achieved in the RP-LC dimension. Detection is performed using UV absorbance. The procedure is validated with respect to recovery, linearity and intra- and interday precision. The lower limit of quantitation (LOQ) is equal for all enkephalins studied and determined to be 5 ng at a signal-to-noise ratio of 5. Based on the injection volume of 5 μl in the first dimension a concentration LOQ of 1000 ng/ml has been established. Although the developed on-line coupled two-dimensional separation system is suited for the quantitation of various enkephalins in the presence of albumin with a satisfactory linearity, precision and recovery, the method lacks still the sensitivity to allow measurements of endogenous enkephalins in a biological matrix. More sensitive detection methods or miniaturization of the system are being tested currently. The value of the present study, however, is that it demonstrates the potency of the two-dimensional system for quantitative purposes in peptide profiling in order to establish the normal peptide profiles of healthy biosystems and to study quantitative modifications of these profiles in case of diseases.