Page 419 - Multidimensional Chromatography

Forensic and Toxicological Applications 411

plasma are described here, showing the utility of the first HPLC column to remove
matrix components.
LC–LC has been widely applied for applications such as the determination of
drugs and metabolites from blood. In this case, it is common to use an alkyl-bonded
first column, to allow the proteinaceous material to pass through, while retaining the
drugs of interest. Fouling of the first column with proteins and other high-molecular-
weight interferences is a major problem in this technique. In order to avoid this
problem, size-exclusion chromatography (SEC) is also commonly used as the pre-
column procedure. Again, the large interferences elute rapidly, while the smaller
drug molecules are retained.
Micellar HPLC, developed in the 1970s and 1980s has provided both alternatives
to and interesting methods for multidimensional analysis. Micellar systems have
been used by many workers for on-line sample clean-up, with an emphasis on the
direct injection of biological fluids such as urine or blood. In a typical application,
Posluszny and Weinberger (9) used sodium dodecyl sulfate above the critical micelle
concentration as a mobile phase modifier. The micelles form a pseudo-stationary
phase, which moves along with the solvent flow. This third phase can effectively par-
tition interfering components, thus removing them from the column and avoiding
fouling of the stationary phase. In multidimensional applications, the second column
can mitigate the well-known efficiency limitations of single-dimension micellar
chromatography. When the process is automated by the use of switching valves,
automated, on-line enrichment and separation of drugs from complex urine and
blood samples can be performed. Posluszny and Weinberger (9) analyzed several
drugs from plasma, with direct injection of the plasma onto their multidimensional
system. It is worth noting that they employed a ‘consumable’ inexpensive pre-
column, which they simply replaced when fouled, typically after several hundred
injections. In Figure 15.3, an analysis of tricyclic antidepressants from blood plasma
is shown. Filtration was the only sample treatment prior to injection and no carry-
over between injections was seen. It is seen that excellent efficiency and separation
of the drug from the matrix can be achieved in a relatively short (less than 20 min)
analysis time. These authors also demonstrated the direct injection and identification
of drugs in plasma by using multidimensional high performance liquid chromatogra-
phy (HPLC) with photodiode array detection (10).
More recently, Carda-Broch et al. described the analysis of cardiovascular drugs
from urine by using micellar HPLC (11). These authors present extensive method
development information, including resolution maps showing micelle and mobile
phase composition. In effect, this may be viewed as a multidimensional technique
within a single column. Typical separations of beta-blockers, using three different
mobile phase compositions, are shown in Figure 15.4. Although analysis times are
somewhat long, excellent resolution can be obtained, and tremendous effects on the
selectivity are seen.
Stopher and Gage used size-exclusion chromatography (SEL) coupled to reversed
phase HPLC for the direct injection of plasma in the analysis of an antifungal agent,
voriconazole (12). Their system consisted of three columns, i.e. first a size-exclusion