A
direct HPLC metho
d was
develope
d for the enantioseparation of pantoprazole using macrocyclic glycopepti
de-base
d chiral stationary phases, along with various metho
ds to
determine the elution or
der without isolation of the in
divi
dual enantiomers. In the preliminary screening, four macrocyclic glycopepti
de-base
d chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), an
d teicoplanin-aglycone (Chirobiotic TAG) were screene
d in polar organic an
d reverse
d-phase mo
de. Best results were achieve
d by using Chirobiotic TAG column an
d a methanol-water mixture as mobile phase. Further metho
d optimization was performe
d using a face-centere
d central composite
design to achieve the highest chiral resolution. Optimize
d parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostate
d at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, an
d 0.6 mL/min flow rate. Enantiomer elution or
der was
determine
d using HPLC hyphenate
d with circular
dichroism (CD) spectroscopy
detection. The online CD signals of the separate
d pantoprazole enantiomers at selecte
d wavelengths were compare
d with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapi
d, multiscan CD signals were compare
d with the quantum chemically calculate
d CD spectra. Furthermore,
docking calculations were use
d to investigate the enantiorecognition at molecular level. The molecular
docking shows that the
R-enantiomer bin
ds stronger to the chiral selector than its antipo
de, which is in accor
dance with the
determine
d elution or
der on the column—
S- followe
d by the
R-isomer. Thus, combine
d metho
ds, HPLC-CD an
d theoretical calculations, are highly efficient in pre
dicting the elution or
der of enantiomers.
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