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ZHANG ET AL.
Enantiomers were separated on a cellulose tris-(3,5-dime-
thylphenylcarbamate) (CDMPC)-based chiral stationary
phase (CSP, provided by the Department of Applied Chem-
istry, China Agricultural University, Beijing) under normal-
phase conditions. The CSP was prepared according to the
procedure described in the literature.16,17 CSP was packed
into a 250 3 4.6 mm (i.d.) stainless steel column. The mo-
bile phase was a mixture of 95% n-hexane and 5% n-propanol
with a flow rate of 1 ml/min. Chromatographic separation
was conducted at 208C and UV detection at 220 nm.
Sample Preparations
Substrate-depletion studies in vitro were performed by
incubation of BX (80 lM) or its enantiomers (40 lM) for
rat liver microsomes and BX (60 lM) or its enantiomers
(30 lM) for rabbit liver microsomes with 1 mg microsomal
protein in 50 mM Tris-HCl buffer (pH 7.4) with 5.0 mM
MgCl2. BX or its enantiomers were prepared in alcohol
and added to incubations. The volume of alcohol added to
each incubate was less than 1.0% v/v. All reaction mixtures
were preincubated in a heated water bath at 378C for
5 min before initiation of the reaction with the addition of
NADPH at a final reaction concentration of 1.0 mM, the
final total reaction volume was 1.0 mL. After incubation in
a water bath (378C) for 5–40 min, the reactions were termi-
nated by adding 5 mL of ice-cold ethyl acetate, the sample
was vortexed for 5 min. After centrifugation at 3500 rpm
for 5 min, the clear solution was decanted into a test tube.
The extraction and centrifuge steps were repeated with
another 5 mL of ethyl acetate. The organic phase was com-
bined and evaporated to dryness under a stream of nitro-
gen at 508C, the resulting residue was redissolved in
200 ll 2-propanol for HPLC analysis.
Fig. 1. Chemical structure of benalaxyl (BX) enantiomers.
HPLC with a preparative chiral column (cellulose-tris-(3,5-
dimethylphenylcarbamate)-based chiral stationary phase;
CDMPC-CSP, provided by the Department of Applied
Chemistry, China Agricultural University, Beijing). Water
was purified by a Milli-Q system. Stock solution of racemic
standard was prepared in 2-propanol and was stored at
2208C. Working standard solutions were obtained by dilu-
tions of the stock solution in 2-propanol. b-Nicotinamide
adenine dinucleotide phosphate (NADPH) was purchased
from Sigma-Aldrich (St Louis, MO, USA). n-propanol,
n-hexane, and ethyl acetate (analytical grade) were from
Yili Fine Chemicals (Beijing, China), distilled and filtered
through a 0.45 lm filter membrane before use. All other
chemicals and solvents were analytical grade and pur-
chased from commercial sources.
Preparation of Rat and Rabbit Liver Microsomes
Animals were sacrificed and the liver was quickly
removed, blotted, weighed, and placed in ice-cold 1.15%
KCl solution. Tissue was minced with scissors and washed
with 1.15% KCl solution to remove blood. After draining
the 1.15% KCl solution, individual liver was homogenized
in ice-cold SET solution (1 mM ethylenediamine tetra-ace-
tic acid and 50 mM Tris-HCl, pH 7.4). The homogenate
was centrifuged at 10,000g for 20 min at 48C and the pellet
was discarded. The supernatant was centrifuged at
108,000g for 60 min at 48C. The supernatant (cytosol) was
decanted. The pellet was washed with 50 mM Tris-HCl
and the homogenate was centrifuged at 108000g for 60
min at 48C again. The pellet was resuspended in 50 mM
Tris-HCl buffer (pH 7.4) containing 20% glycerol. This pro-
cedure was used for the preparation of microsomes from
Sprague-Dawley rat (male, n 5 6) and Japanese white rab-
bits (male, n 5 3). Protein concentration was determined
by the method of Bradford with BSA as the standard,8 and
microsomes were stored at 2808C until used.
Data Analysis
The enantiomer fraction (EF) was used to measure the
enantioselectivity of the BX enantiomers. EF of (2)-R-BX
is defined by eq. 1.EF 5Peak areas of the
ðꢀÞ
EF ¼ Peak areas of the
ð1Þ
½ðþÞ þ ðꢀÞꢁ
where (2) and (1) are the first and second enantiomers
determined by the result of polarimeter in previous
works.11 The EF for racemate is 0.50, whereas preferential
degradation of the (1) or (2) yields EF <0.50 and >0.50,
respectively.
The metabolism of rac-BX or its enantiomers appeared
to follow a first-order kinetic reaction, and the degradation
rate constants were derived from ‘‘ln(C0/C) versus t’’ plots
by regression analysis for experiment (Excel 2003, Micro-
soft1). The starting point was the maximum concentra-
tion. The in vitro elimination half-life (t1/2) was determined
by the following eq. 2:
HPLC-DAD Analysis
Chromatography was performed using an Agilent 1100
series HPLC equipped with a G1311A pump, G1322A
degasser, G1328A injector, a 20-ml sample loop, and
G1315A DAD. The signal was received and processed by
an Agilent Chemstation for 3D LC (Agilent Technologies,
Palo Alto, CA).
0:693
t1=2
¼
ð2Þ
k
Nonlinear regression of substrate concentration versus
reaction velocity curves were analyzed using Origin 7.5
Chirality DOI 10.1002/chir
Zhang, Ping
