Page 275 - Multidimensional Chromatography

270 Multidimensional Chromatography

after the injection of 200 serum samples of 500 l. An on-line sample clean-up using
a Pinkerton-GFF2 RAM pre-column was combined with LC on a non-porous ODS
phase for the analysis of six cardiovascular drugs in serum (97). Matrix interferents
could be removed efficiently and were not detected in the serum chromatograms.

11.3.3 IMMUNOAFFINITY SORBENTS IN SPE–LC

In the large majority of all SPE–LC applications, SPE pre-columns are packed with
a hydrophobic packing material such as an ODS phase or a polystyrene–divinyl-
benzene copolymer. With these packing materials, in principle, sensitivity can be
increased dramatically (high analyte retention), but selectivity may only be slightly
improved (non-selective hydrophobic interaction). If enhanced selectivity is a
major requirement, packing materials with immobilized antibodies (immunoaffinity
sorbents) can be used which provide extraction based on molecular recognition.
After some exploration work of others (99, 100), Farjam and co-workers were the
first to extensively study the on-line application of immunoaffinity pre-columns in
combination with LC for the selective determination of low-molecular-weight com-
pounds (steroids and aflatoxins) in biological samples (101–105). A system con-
sisting of a pre-column packed with Sepharose-immobilized polyclonal antibodies
against -19-nortestorenone ( -19-NT), a second ODS-filled pre-column, an ana-
lytical ODS column and a UV detector (Figure 11.7(a)), was used for the determi-
nation of -19-NT and -19-NT in calf urine which was injected directly (101).
The analytes were subsequently trapped on the immunoaffinity pre-column, selec-
tively desorbed by using the cross-reacting steroid norgestrel, reconcentrated on the
ODS precolumn and transferred to the analytical column. Recoveries of -19-NT
were over 95% and the detection limit was 50 ng/l for a 25 ml urine injection
(Figure 11.7(b)). A similar procedure was also used for the determination of -19-
NT and -19-NT in calf bile, liver, kidney and meat samples (102), and for the anal-
ysis of clenbuterol in calf urine (106). In a subsequent study, an anti-aflatoxin
immunoaffinity pre-column was used on-line for the selective preconcentration of
aflatoxin M1 from urine and milk samples (103, 104). By using LC with fluores-
cence detection following the immuno-SPE step, detection limits of 20 ng/ml for
aflatoxin M1 were found if 2.4-ml milk samples are used. The lifetime of the
immunoaffinity pre-column was strongly reduced when milk samples were anal-
ysed, due to proteolic enzymes in milk which degrade the antibodies. This problem
was circumvented by adding an on-line dialysis unit to the system to prevent inter-
fering macromolecular compounds from entering the pre-column (104). A single
immunoaffinity pre-column could now be used for over 70 milk analyses. Another
example of the use of on-line immunoaffinity-SPE–LC is the selective quantifica-
9
tion of -tetrahydrocannabinol, the major indicator of cannabis intoxication, in
human saliva (107).
Immunoaffinity extraction combined on-line with LC in conjunction with MS
(108–110) or tandem MS (111, 112) has also been demonstrated for the determina-
tion of analytes in biological fluids. Obviously, such systems offer a very high