ZHU ET AL.
3
Among HPLC methods for enantioseparation, the use
Shenyang Pharmaceutical University, Shenyang, China.
Two synthetic routes and the corresponding final
products are shown in Figure 1. Methanol (MeOH),
acetonitrile (ACN), CH2Cl2, THF, and n‐hexane of HPLC
grade were purchased from Concord Technology Co. Ltd.
(Tianjin, China). Isopropyl alcohol (IPA) and ethanol
(EtOH) of HPLC grade were purchased from Yuwang
Industrial Co. Ltd. (Shandong, China).
of CSPs has become the most popular tool for determina-
tion of the enantiomeric impurities of wide number of
chiral drugs, as it is rapid, reproducible, and effective.9
The choice of CSP is certainly the dominant factor
contributing to successful separation. Numerous CSPs
are commercially available, and the polysaccharide‐based
CSPs in the Chiralpak and Chiralcel series have been the
most widely utilized materials for enantiomeric resolution
by liquid chromatography.10 Polysaccharide‐based CSPs
are produced either by physical coating or immobilization
of the chiral polymers on silica support. However, the
coated CSPs can only be used with a rather limited eluent,
because some commonly used solvents, such as chloro-
form, acetone, and tetrahydrofuran (THF), can swell or
dissolve the polysaccharide derivatives on silica gel.
However, the use of these prohibited solvents may be
useful for resolving recemates, which could not be
separated by using low‐polarity solvents.11,12 To enhance
the solvent compatibility for these CSPs, the immobilized
ones have been commercialized, which possess
advantages in CSP robustness and extended range of
solvents and applications.13,14 In addition, the coated
CSPs could be used either under normal phase or reversed
phase, while the immobilized CSPs can be used in both
modes.
2.2 | Apparatus
All chromatographic measurements were performed on an
HPLC system consisting of a PU‐1580 pump (Jasco, Tokyo,
Japan) and SPD‐15C UV‐Vis detector. Data acquisition
was performed by using a N2000 Chromatography Data
System obtained from Zhida Information Engineering
Co. Ltd. (Zhejiang, China). The chiral columns, Chiralpak
AD‐H (250 × 4.6 mm), Chiralcel OD‐RH (250 × 4.6 mm)
and Chiralpak IC (250 × 4.6 mm) were obtained from
Daicel Chiral Technologies (China) Co., Ltd, Shanghai,
China. The chiral selectors of these CSPs were tris
(3,5‐dimethylphenylcarbamate) of amylose coated on
5 μm silica gel, tris(3,5‐dimethylphenylcarbamate) of
cellulose coated on 5 μm silica gel, and tris(3,5‐
dichlorophenylcarbamate) of cellulose immobilized on
5 μm silica gel, respectively.
In the present study, attempts have been made to
resolve Ez stereoisomers on both coated and immobilized,
normal and reversed phase CSPs, namely, Chiralpak
AD‐H, Chiralcel OD‐RH, and Chiralpak IC. For the
coated CSP, Chiralpak AD‐H was used in normal phase
mode with mixtures of hexane and alcohol as mobile
phases, and Chiralpak OD‐RH was applied in the
reversed‐phase separation with aqueous mobile phases.
But for Chiralpak IC, as the immobilized CSP, we
uniquely tested unusual mobile phase compositions
containing mixtures of hexane and THF or CH2Cl2 for
separation. This study aimed to explain the separation
results of Ez stereoisomers on various stationary phases
and also the effects of different mobile phases on the
enantioselectivity and resolution in normal and reversed
phase modes. Based on the enantioseparation, the most
suitable CSP and mobile phase were selected for the
quantitative determination of (RRS)‐Ez in Ez bulk drug.
2.3 | Chromatography
Various mobile phase systems were investigated for the
HPLC study. All of them were composed of commonly
used organic HPLC solvents and water. The method
validation was carried out on a Chiralpak IC column
(250 × 4.6 mm id, 5 μm, Daicel Chiral Technologies Co.,
Ltd., Shanghai, China) with a mobile phase consisting of
hexane/IPA (90/10, v/v) at a flow rate of 1.0 mL min−1
.
The injection volume was 20 μL, and the detection
wavelength was set at 232 nm. The temperature was kept
at 25°C except where the effect of temperature was
studied. An amount of 2 mg each Ez and (RRS)‐Ez was
dissolved in 10 mL of mobile phase. The resultant solution
had 0.2 mg mL−1 of individual concentration. The target
analyte concentration of Ez was fixed as 0.2 mg mL−1. A
0.2 mg mL−1 solution of Ez prepared in mobile phase,
which was spiked with 1 μg mL−1 (RRS)‐Ez, was utilized
as standard spiked solution.
All the chromatographic parameters given in this
work were the mean values from 3 consecutive injections.
Retention factor (k′) was calculated from (tR − t0)/t0,
where t0 was the void time estimated from the earliest
baseline perturbation and tR was the retention time of
enantiomeric solute. Separation factor (α) was calculated
from k′2/k′1, where k′1 and k′2 were the retention factors
2 | MATERIALS AND METHODS
2.1 | Chemical and reagents
Ezetimibe, (RRS)‐Ez, (RSR)‐Ez, and (SSR)‐Ez (purity>99.0%) were
purchased from Nanjing Healthnice Pharmaceutical Co.
Ltd. (Nanjing, China). The mixtures of stereoisomers were
synthesized by School of Pharmaceutical Engineering,