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extracts of human lung and liver tissues. Matsuoaka combined anion-exchange LC
with RPLC for the analysis of the enzymatic digests of bovine calmodulin and D59
protein, and for the separation of a crude peptide mixture which was extracted from
bovine brain tissue.
With comprehensive two-dimensional LC, indeed truly orthogonal separations
with very high efficiency and peak capacity can be obtained, although the interfacing
can be quite complicated. Additionally, in bioanalysis large peak capacities are often
not needed, especially when the objective is the determination of only a few target
compounds (see next section). The usefulness of comprehensive LC–LC lies partic-
ularly in the entire profiling of protein and peptide mixtures of unknown composi-
tion (protein/peptide mapping). In this respect, comprehensive LC–LC might be an
alternative for two-dimensional gel electrophoresis, with LC–LC having the advan-
tage that it can be on-line coupled to MS so that molecular-weight information
becomes available fast and without the need for analyte transfer.

11.2.2 HEART-CUT LC–LC

In contrast to comprehensive LC–LC, the goal of the targeted mode of multidimen-
sional LC is to isolate a single analyte or small group of components of a complex
mixture. Target analysis by LC–LC is normally carried out by using the principle of
‘heart-cutting’. In this approach, an eluting zone from the first LC column (heart-cut)
is switched to a second column for subsequent analysis of the transferred com-
pounds. Thus, the first column is used to extract and enrich analytes, e.g. from com-
plex biological fluids, while the second column is used to separate the molecule(s) of
interest. This combination leads to an enhanced selectivity and sensitivity, because
the target compounds are preconcentrated and/or can be detected in the absence (or
reduced presence) of interfering compounds. As a result, lower detection limits are
obtained and the reliability of quantitative analysis is strongly improved. When a
very high selectivity is required, the dimensionality (number of subsequent columns)
may be increased to three or more by using different retention modes. Of course,
on-line LC–LC requires the use of mobile phases which, to some extent, are com-
patible with the columns involved. Therefore, LC–LC is restricted to certain chro-
matographic combinations, which often include RPLC, ion-exchange, polar
bonded-phase, chiral or affinity columns. It is evident that next to the selection of the
proper columns and eluents, correct timing of the column switching and optimiza-
tion of the transfer volume are essential aspects, and, therefore, systematic method
development is an important issue in LC–LC (13–15).
In biomedical analysis, LC–LC has been used most extensively and successfully
in the heart-cut mode for the analysis of drugs and related compounds in matrices
such as plasma, serum or urine. Table 11.1 gives an overview of analytes in biologi-
cal matrices which have been determined by heart-cut LC–LC systems. A typical
example of such an approach is the work of Eklund et al. (16) who determined the
free concentration of sameridine, an anaesthetic and analgesic drug, in blood plasma