Page 270 - Multidimensional Chromatography

Biomedical and Pharmaceutical Applications 265

Considering the numerous applications, heart-cut LC–LC has convincingly
proven its value. Nevertheless, in LC–LC specific method development is generally
needed for each analyte. Moreover, heart-cut procedures require accurate timing
and, therefore, the performance of the first analytical column in particular should be
highly stable to thus yield reproducible retention times. This often means that in
LC–LC some kind of sample preparation remains necessary (see Table 11.1) in
order to protect the first column from proteins and particulate matter, and to guaran-
tee its lifetime.

11.3 SOLID-PHASE EXTRACTION–LIQUID CHROMATOGRAPHY

Today, solid-phase extraction (SPE) is probably the most popular sample preparation
method used for sample enrichment and/or clean-up (3). During the last decade,
many improvements in the SPE field such as new formats (e.g. sophisticated car-
tridges and discs, pipette tips and 96-well plates), new sorbents and the development
of automated systems, have led to an extensive use of SPE. Moreover, the on-line
combination of SPE and LC using column switching techniques is now routine. SPE,
can in principle, be described as a simple liquid chromatographic process in which
the sorbent is the stationary phase. Therefore, the direct coupling of SPE and LC can
be considered to be a multidimensional chromatographic technique. However, in
contrast to LC–LC, in SPE–LC no continuous elution is carried out during the first
dimension. Ideally, the analytes are fully retained on the SPE phase during the sorp-
tion and washing steps, and, subsequently, quickly and completely desorbed in a
small volume during the elution step. In an on-line SPE–LC set-up the desorption
solvent is entirely injected on to the LC column.
Numerous studies involving the on-line coupling of SPE columns to LC analyti-
cal columns have been reported, including a significant number of biomedical appli-
cations (70). Various drugs and endogenous compounds have been analysed in
biological matrices by employing this technique, including antibiotics (71), retinoids
(72, 73), methotrexate (74), codenie (75), aspirin (76), psilocin (77), almokalant
(78), anabolic steroids (79), morphine (80), ceftazidime (81), terbinafine (82), cloza-
pine (83), mizolastine (84), cebaracetam (85) and piroxicam (86). In many of these
applications ODS sorbents are used for SPE. Typically, 0.1–1 ml of plasma or ca.
1–10 ml of urine are loaded on a preconditioned SPE cartridge (2–15 mm long,
1–4.6 mm i.d., packed with 15–40 m particles) which is mounted on a six- or ten-
port valve and replaces the conventional injection loop. After washing (clean-up),
the pre-column is desorbed with mobile phase which is led to the analytical column
(often packed with an RPLC phase) for separation of the analytes. Quantification
and/or identification is frequently carried out using UV or MS detection. During
analysis, the SPE cartridge may be cleaned, reconditioned and loaded with the next
sample. Figure 11.5 shows a typical example of on-line SPE–LC for the determina-
tion of drugs in human plasma (87). Many of the on-line SPE–LC applications for
bioanalysis described in the literature are quite similar and we will not further dis-
cuss these studies. In the remaining part of this section, some selected at-line and