Biomedical Engineering – From Theory to Applications

Column Coupling Electrophoresis in Biomedical Analysis
Fig. 15. ITP-EKC-DAD method for the direct sensitive determination of enantiomers in
unpretreated complex matrices sample with spectral characterization of electrophoretic
zones. 3D traces were obtained combining electrophoretic (EKC) and spectral (DAD) data
where the spectra were scanned in the interval of wavelengths 200-400 nm. (a) 3D trace
illustrating the whole EKC enantioseparation of pheniramine and its metabolites in the online
pretreated clinical urine sample (spectra of matrix constituents, well separated from the
analytes, are pronounced), (b) detail on the 3D spectra showing the migration positions of
pheniramine enantiomers (E1 and E2) and their structurally related metabolites (M1 and
M2). The spectrum of the little unknown peak marked with the asterisk differed from the
pheniramine spectrum significantly and, therefore, it was not considered as a pheniramine
biodegradation product. The urine sample was taken 8.5 hours after the administration of
one dose of Fervex (containing 25 mg of racemic pheniramine) to a female volunteer and it
was 10 times diluted before the injection. The separations were carried out using 10 mM
sodium acetate - acetic acid, pH 4.75 as a leading electrolyte (ITP), 5 mM -aminocaproic
acid - acetic acid, pH 4.5 as a terminating electrolyte (ITP), and 25 mM -aminocaproic acid -
acetic acid, pH 4.5 as a carrier electrolyte (EKC). 0.1% (w/v) methyl-hydroxyethylcellulose
served as an EOF suppressor in leading and carrier electrolytes. Carboxyethyl--CD (5
mg/mL) was used as a chiral selector in carrier electrolyte. Reprinted from ref. (Marák et al.,
2007), with permission.
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