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
T. Stroink et al., 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, ANALYT CHIM, 444(2), 2001, pp. 193-203
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.
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