Page 273 - Multidimensional Chromatography

268 Multidimensional Chromatography

detector was applied for the analysis of omeprazole, a drug for the treatment of gas-
tric ulcers, in human plasma (87). The extraction was carried out on-line by using
disposable SPE cartridges filled with an ODS sorbent, which, after washing, yielded
sufficient sample clean-up and enrichment to permit measurement of drug levels at
the low ng/ml level (see Figure 11.5). The automated system was validated and used
for a large number of plasma samples from bioequivalence studies. In another study,
adapalene (a retinoid) and retinol were simultaneously determined in human plasma
and mouse tissue by combining an autosampler and an OSP-2 unit with a gradient
LC with UV and fluorescence detection (92). The high degree of automation of the
on-line SPE–LC system, its high sensitivity (LOQ of 0.25 ng/ml for adapalene) and
its good reproducibility, made this method convenient for the determination of phar-
macokinetic drug profiles.

11.3.2 RESTRICTED-ACCESS SORBENTS IN SPE–LC

In on-line SPE–LC, deproteination of plasma and serum is often required before
extraction, especially when the same SPE cartridge has to be used for repeated anal-
yses. However, today there is strong interest in on-line sample pretreatment tech-
niques which permit the handling of untreated biological samples. For this purpose,
special SPE sorbents (so-called restricted-access materials (RAMs)) have been
developed which allow sorption (enrichment) of low-molecular-weight analytes at
the inner pore surface of the particles, but at the same time exclude high-
molecular-weight matrix compounds (e.g. proteins) from the pores (Figure 11.6(a)).
In recent years, RAMs have become quite popular for the direct injection of biologi-
cal fluids into on-line SPE–LC systems (93–98). For instance, Yu and Westerlund
(93) selected a RAM pre-column filled with an alkyl-diol silica to rapidly separate
bupivocaine (a local anaesthetic) from proteins and polar endogenous compounds in
human plasma. A 500 l plasma sample was directly introduced on to the SPE
column and the fraction containing the analyte was on-line transferred to an RPLC
column for final separation. A single alkyl-diol silica pre-column could withstand
over 50 ml of plasma injections. The same type of RAM pre-columns appeared to be
very useful for on-line clean-up and enrichment during the determination of several
drugs and metabolites in biological fluids (e.g. serum, urine, intestinal aspirates and
supernatants of cell cultures) which could be directly injected (94).
LC–MS with on-line SPE using a RAM pre-column with an internal ODS phase
was described by van der Hoeven et al. (95) for the analysis of cortisol and pred-
nisolone in plasma, and arachidonic acid in urine. The samples were injected directly
and the only off-line pretreatment required was centrifugation. By using the on-line
SPE–LC–MS system, cortisol and related compounds could be totally recovered
and quantified in 100 l plasma within 5 min with a typical detection of 2 ng/ml
(Figure 11.6(b)). The RAM-type of sorbents, in which the outer surface of the parti-
cles is covered with 1 -acid glycoprotein, also appear to be useful for direct SPE of