Biomedical Engineering Reference
In-Depth Information
investigation of CNS penetration in a rat in vivo study. Unfortunately, recovery was
not reported.
Whereas examples described above are characterized by separated steps of sam-
ple extraction and subsequent separation by the HPLC system, Boppana et al. pre-
sented a quick online automated sample enrichment design that allowed direct
injection of plasma samples with immediate consecutive chromatographic separa-
tion for MRM analysis of granisetron (Fig.
1
) and its biotransformation products
[
100
]. Following this approach plasma was directly flushed onto a guard column
packed with restricted access media (RAM) characterized by an internal surface
reversed-phase (ISRP). RAM is a special porous extraction sorbent with dual prop-
erties caused by its non-adsorptive outer hydrophilic surface and its adsorptive
internal surface. It exhibits both a sieving effect (size exclusion mechanism) thus
preventing large proteins from entering the small pores of the sorbent and an adsorp-
tion effect (often reversed-phase) to small molecules that have entered the pores.
Therefore, plasma proteins and other large matrix components immediately pass
through the guard column without clogging whereas small molecule analytes are
retained on the inner surface. Switching the flow direction of an appropriate solvent
(reverse to injection) allows elution of the analytes, which are directly transferred
onto the separation column for analysis.
A similar approach with multiple columns operating as automated multi-dimen-
sional LC has also been reported by Machtejevas et al. for peptides from human
blood fi ltrate [
101
] .
All examples for sample preparation procedures discussed above were followed
by LC-MS or LC-MS/MS analysis of TTA and QTA. The next sections are addressed
to the typical performance of sample separation and analyte detection.
3.2
Chromatographic Separation
Reliable chromatographic methods for LC-MS have to achieve efficient separation
of multiple analytes or interfering matrix components by using solvents that allow
compatibility to MS detection. Therefore, HPLC solvents should not contain inor-
ganic salts as typical for ion pair or ion exchange chromatography as they might
cause clotting of the MS entrance. Optimum prerequisites for coupling to MS are
provided by reversed-phase chromatography separating compounds on a hydropho-
bic stationary phase (C
8
-C
18
, phenyl) in combination with an aqueous mobile phase
containing an appropriate ratio of organic modifier (e.g. ACN, MeOH) that elutes
the analytes.
3.2.1
Reversed-Phase Chromatography
Nearly without exception TTA and QTA were separated on RP-material as summa-
rized in Tables
5
-
8
. In practice, the choice of a specific RP-column often depends
