5
0
ZAAZAA ET AL.
membrane filter followed by degassing with ultrasonic vibration prior to
EXPERIMENTAL
Apparatus
use. The injection volume was 20 μL. The column was conditioned for
at least 30 min. The detection was achieved with UV detection at 210
nm and 225 nm for hyoscyamine sulfate and eszopiclone, respectively.
The flow rate was 0.8 and 0.5 mL/min for hyoscyamine sulfate and
zopiclone, respectively.
Analytical chromatography was performed on an HPLC system: Agilent
Technologies 1200 series (Germany), consists of an Agilent 1200 series
variable wavelength detector G1314B/G13145 (SL), Agilent 1200 series
vacuum degasser (20 μL), Agilent 1200 series manual injector, and
Agilent 1200 series quaternary pump G1310 A, G1311 A. Agilent Syringe,
LC 50 μL, (USA) was used for injection. The column was heated and
cooled by an Agilent temperature controller system B558. Chiral AGP col-
umn, 150 × 4.0 mm, 5 μm (ChromTech, Apple Valley, MN) was used for
separation. Cellulose acetate filter papers, dimension 47 mm, pore size
Screening approach of direct chiral AGP method. Following the
5
method development protocol for chiral separation on an AGP column,
different mobile phases were tried including mixtures of phosphate or ac-
etate buffers and organic modifiers, such as isopropanol or acetonitrile.
The enantioselectivity and retention can easily be regulated by mobile
phase, pH and ionic strength, and the nature and concentration of the or-
ganic modifier. The most important tool in method development is the
mobile phase pH and the ionic strength, which affect the ionization of
both solutes.
0
(
.45 μm (ChromTech) was used for filtration. An ultrasonic sonicator
Memmert-Germany) was used for degassing.
Samples
Pure samples. Hyoscyamine sulfate was supplied by Sigma Tech for
Pharmaceutical Industries (Giza, Egypt) and its purity was identified as
9.08 ± 0.943. S-(–) hyoscyamine sulfate was supplied by the Egyptian
Group for Pharmaceutical Industries (EGPI, Cairo, Egypt) and its purity
was identified as 99.73 ± 0.369 according to the official USP27 method.
Zopiclone was supplied by Amoun Pharmaceutical Industries (Cairo,
Egypt) and its purity was identified as 100.84 ± 1.944.26 Eszopiclone was
2
6
9
Method Validation28
The method was applied for determination of pure isomers of
eszopiclone and S-hyoscyamine. The developed method was validated
in accordance with ICH guidelines by documenting their linearity, accu-
racy, precision, and limits of detection and quantification.
supplied by Medizen Pharmaceutical Industries (Alexandria, Egypt)
and its purity was identified as 98.76 ± 0.861.26
Application to Drug Products
Market samples. Isopto-atropine ophthalmic solution, 5 mL; labeled to
contain 1 g hyoscyamine sulfate / 100 mL solution, manufactured by
Alcon-Couvreur (Belgium) (Batch No. 240512).
Hypnor tablet; labeled to contain 7.5 mg/tablet of zopiclone,
manufactured by Amoun Pharmaceutical Industries (Batch No. 21559).
For hyoscyamine sulfate, the content of 10 plastic bottles of Isopto-
Atropine ophthalmic solution were mixed and an accurately measured
volume of 1 mL equivalent to 10 mg of hyoscyamine sulfate was directly
transferred into a 100-mL volumetric flask, and diluted to volume with mo-
bile phase. Then the proposed procedure was followed.
For zopiclone, 20 tablets of Hypnor tablets were weighed and ground to a
fine powder. An adequately weighted amount equivalent to 100 mg of
zopiclone was transferred to a 100-mL calibrated volumetric flask and 50 mL
acetonitrile was added. The contents of the flask were sonicated for 20 min
to effect complete dissolution of zopiclone and filtered. The filtrate was quan-
titatively transferred to a 100-mL calibrated volumetric flask and completed to
the volume with acetonitrile. Then the proposed procedure was followed.
Chemicals and Reagents
All reagents used were of HPLC grade and chemicals were of analytical
grade.
Sodium dihydrogen orthophosphate (Qualikems, India), acetonitrile
(Poch, Poland), methanol (Scarlau, Spain), isopropanol, triethylamine,
TEA (Sigma Aldrich, Germany), ethanol (Fischer Scientific, UK), ortho-
phosphoric acid (Lab. Scan, Ireland) were the solvents used.
Water for HPLC was prepared by double glass distillation and filtration
through a 0.45 μm membrane filter.
RESULTS AND DISCUSSION
The optimized mobile phase was found to be, 42.8 mM TEA
in 50 mM sodium phosphate buffer, pH 7, acetonitrile (90: 10
v/v) for hyoscyamine sulfate, and 7.5 mM TEA in 10 mM
sodium phosphate buffer, pH 6, isopropanol (97: 3 v/v)
for zopiclone. The order of elution of hyoscyamine enantio-
mers was S (–) enantiomer then R (+) enantiomer with
retention times of 5.95 and 6.70, respectively. The order of
elution of zopiclone enantiomers was R (+) enantiomer then
S (–) enantiomer with retention times of 6.02 and 7.93, respec-
tively (Figs. 1 and 2). The method was specific for
enantioseparation of the two racemic mixtures of the studied
drugs with baseline separation and resolution of 1.60 and 2.45
for hyoscyamine sulfate and zopiclone, respectively.
Standard Solutions (100 μg/mL)
An accurately weighed amount, 10 mg of each active isomer of hyoscy-
amine sulfate, eszopiclone, and racemic mixtures of hyoscyamine sulfate
and zopiclone were transferred separately into 100-mL volumetric flasks
and dissolved in 20 mL mobile phase, and then the volumes were com-
pleted with mobile phase (100 μg/mL).
Working standard solutions. Working standard solutions were pre-
pared in concentration ranges of 2–20 μg/mL hyoscyamine sulfate and
–10 μg/mL hyoscyamine sulfate, 1–10 μg/mL zopiclone, and 0.5–5
1
μg/mL eszopiclone by appropriate dilution of standard solution with mo-
bile phase.
Stability of solutions. The solutions were stable for about 1 week
when stored at 4°C, and no evidence of degradation of the analytes was
observed on the chromatograms during this period.
Screening Approach of Direct Chiral AGP Method
The effect of individual variables: buffer, pH, and type of
modifier were investigated according to the AGP protocol.
The results of the screening study are presented in Table (1).
Procedures
Chromatographic conditions. All chromatographic experiments were
carried out in the isocratic mode. HPLC was carried out at 10°C on a Chi-
ral AGP column, 150 × 4.0 mm, 5 μm. The mobile phase consisted of 42.8
mM TEA in 100 mM sodium phosphate buffer: acetonitrile (90: 10, v/v)
pH 7 for hyoscyamine sulfate, and 7.5 mM TEA in 10 mM sodium phos-
phate buffer pH 6 (adjusted with o-phosphoric acid): isopropanol (97: 3,
v/v) for zopiclone. The mobile phases were filtered using a 0.45 μm
Chirality DOI 10.1002/chir
Influence of mobile phase pH on enantioselectivity; retention and
selectivity. The influence of mobile phase pH on retention
and selectivity of hyoscyamine sulfate and zopiclone on
chiral AGP stationary phase was studied at pH 3.0 to 7.0,
keeping the concentration of uncharged modifier (10% aceto-
nitrile and 3% isopropanol) and molarity of buffer (100 mM