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Biomedical and Pharmaceutical Applications 277

11.4.2 RPLC–GC

For pharmaceutical and biomedical analysis, RPLC is much more important than
NPLC. However, the interfacing techniques used in NPLC–GC do not generally
work well when used for RPLC–GC. The main difficulties encountered when trans-
ferring water or water-containing eluents to a GC unit are due to the large vapour
volume of water, its high surface tension, poor wetting characteristics, high boiling
point and aggressive hydrolytic reactivity. Two approaches have been described for
interfacing RPLC and GC on-line, i.e. (i) direct introduction of the aqueous LC frac-
tion by miniaturization of the LC step or by use of special retention gaps, and (ii)
phase-switching techniques, i.e. the analytes are first transferred to an organic sol-
vent and subsequently introduced into the GC system.
For drug analysis, Goosens et al. (123) used a Carbowax-deactivated retention
gap to transfer eluents from the LC unit to the GC part of the system. Up to 200 l of
eluent (acetonitrile-water) were introduced into a Carbowax-coated retention gap by
using an on-column interface and solvent vapour exit (124). It was found that the
water content of the eluent should not exceed that of the azeotropic mixture, or
otherwise water, which is left in the gap after evaporation of the azeotropic mixture,
will mar the analysis. In order to deal with the presence of buffers or ion-pairing
agents, an anion-exchange micromembrane device was inserted between the LC and
GC parts of the system to remove the ion-pairing agent methanesulphonic acid from
an acetonitrile–water LC eluent (125). The applicability of the on-line LC–
micromembrane–GC system was illustrated for the potential drug eltroprazine (125)
and for an impurity profile of the drug mebeverine (126). Before the LC fraction was
introduced on-line into the GC–MS system, acetonitrile was added to achieve an
azeotropic acetonitrile/water ratio, and, therefore only a part of the LC peak could be
transferred. Nevertheless, electron impact and chemical ionization spectra of an
impurity could be obtained at a level of 0.1 % with respect to the drug.
Ogorka et al. (127) have coupled RPLC and GC via on-line liquid–liquid extrac-
tion of the aqueous mobile phase and used the system for the impurity profiling of
drugs. The instrumental set-up is shown in Figure 11.11, where a main critical part is
the phase separator. These authors optimized the extraction for mobile phases con-
sisting of methanol–water and acetonitrile–water by using n-pentane, n-hexane and
dichloromethane as extraction solvents. The extraction yield depended on the water
content of the mobile phase and the polarity of the organic phase. Transfer volumes
of 500 l of aqueous mobile phase have been used. The usefulness was extended via
on-column derivatization by the introduction of a reagent via the loop-type interface
or by derivatization during the extraction (128). By the use of MS as the GC detector,
the identification of various unknown impurities in pharmaceutical products was
achieved. Contrary to direct LC–MS, the composition of the LC eluent is less lim-
ited because non-volatile buffers can also be chosen. The same system was also
applied for the analysis of biological samples, i.e. the determination of -blockers in
human serum and urine (129) and the determination of morphine and its analogues
in urine (130). In the latter case, the analytes were silylated with bis (trimethylsilyl)