Máté Dobó, Gergely Dombi, István Köteles, Béla Fiser, Csenge Kis, Zoltán-István Szabó and Gergő Tóth
Abstract
A reversed-phase high-performance liquid chromatographic (HPLC) method was developed
for the simultaneous determination of the potential impurities of dexketoprofen, including the
distomer R-ketoprofen. After screening the separation capability of four polysaccharide columns (Lux
Amylose-1, Lux Amylose-2, Lux Cellulose-1 and Lux Cellulose-2) in polar organic and in reversedphase
modes, appropriate enantioseparation was observed only on the Lux Amylose-2 column in
an acidified acetonitrile/water mixture. A detailed investigation of the mobile phase composition
and temperature for enantio- and chemoselectivity showed many unexpected observations. It was
observed that both the resolution and the enantiomer elution order can be fine-tuned by varying the
temperature and mobile phase composition. Moreover, hysteresis of the retention times and enantioselectivity
was also observed in reversed-phase mode using methanol/water mixtures on amylose-type
columns. This could indicate that the three-dimensional structure of the amylose column can change
by transitioning from a polar organic to a reversed-phase mode, which affects the enantioseparation
process. Temperature-dependent enantiomer elution order and rare enthalpic/entropic controlled
enantioseparation in the operative temperature range were also observed in reversed-phase mode.
To find the best methodological conditions for the determination of dexketoprofen impurities, a
full factorial optimization design was performed. Using the optimized parameters (Lux Amylose-2
column with water/acetonitrile/acetic acid 50/50/0.1 (v/v/v) at a 1 mL/min flow rate at 20 °C),
baseline separations were achieved between all compounds within 15 min. Our newly developed
HPLC method was validated according to the current guidelines, and its application was tested on
commercially available pharmaceutical formulations. According to the authors’ knowledge, this is
the first study to report hysteretic behavior on polysaccharide columns in reversed-phase mode.
Keywords: chiral separation; chiral switch; HPLC; ketoprofen; method optimization; polysaccharide
chiral column; retention curve; thermodynamic