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

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 m ono-dimensional separation systems is not sufficient. In order to improve t he separation efficiency two- or multi-dimensional systems can be the solut ion. The present study aims at the development and validation of an on-line two- dimensional separation system, using size exclusion chromatography (SEC) an d reversed-phase liquid chromatography (RP-LC), to quantitate structurally related peptides in the presence of large proteins such as albumin in contr ast with the available coupled-column systems which are focussed on the qua litative determination of peptides. As model peptides a number of enkephali ns 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 chose n properly, the entire peak, eluting from the SEC column and containing the enkephalins, can be trapped in a loop after complete separation from album in. Subsequent separation of the enkephalins is achieved in the RP-LC dimen sion. Detection is performed using UV absorbance. The procedure is validated with respect to recovery, linearity and intra- a nd interday precision. The lower limit of quantitation (LOQ) is equal for a ll enkephalins studied and determined to be 5 ng at a signal-to-noise ratio of 5. Based on the injection volume of 5 mul in the first dimension a conc entration 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 albu min 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, howe ver, 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 modificat ions of these profiles in case of diseases. (C) 2001 Elsevier Science B.V. All rights reserved.