Page 420 - Multidimensional Chromatography

412 Multidimensional Chromatography

Figure 15.3 Separation of tricyclic antidepressants by using multidimensional LC–LC.
Peak identification is as follows: DOX, doxepin; DES, desipramine; NOR, nortryptylene; IMI,
imipramine; AMI, amitryptyline. Adapted from Journal of Chromatography, 507, J. V.
Posluszny et al., ‘Optimization of multidimensional high-performance liquid chromatography
for the determination of drugs in plasma by direct injection, micellar cleanup and photodiode
array detection’, pp. 267–276, copyright 1990, with permission from Elsevier Science.

column, primarily used for de-salting followed by a pre-column, followed by a C 18
analytical column. The pre-column was used for a final clean-up, plus re-focusing of
the analytes prior to separation on the analytical column. This system allowed the
direct injection of approximately 0.5 mL of plasma. The limit of quantitation of the
method was found to be 5 g/mL, with quantitation by internal standard. Figure 15.5
shows an analysis of voriconazole with an internal standard. Resolution between
these very similar compounds is excellent, with a relatively short analysis time and
little matrix interference.
Another system based on an SEC pre-column was employed by Earley and Tini
for the analysis of a nonpeptidic inhibitor of human leukocyte elastase as part of a
drug discovery project (13). The analytical column used here was octadecylsilane,
although the authors commented that, depending on the particular application, any
stationary phase could be used as the analytical column. In their application, a frac-
tion of the effluent from the SEC column was transferred to the analytical column by
the use of a C 18 collection column that served to pre-concentrate and focus the ana-
lytes. They noted that frequent solvent flushing was required to avoid build-up of
impurities on the collection column. A chromatogram obtained from a 50 L plasma
standard injection is shown in Figure 15.6. This separation provides an excellent