Liquid chromatographic resolution of amino acid esters of acyclovir including racemic valacyclovir on crown ether-based chiral stationary phases

Article abstract of DOI:10.1002/chir.22424

Valacyclovir, a potential prodrug for the treatment of patients with herpes simplex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs) based on (3,3′-diphenyl-1,1′-binaphthyl)-20-crown-6 covalently bonded to silica gel. In order to find out an appropriate mobile phase condition, various mobile phases consisting of various organic modifiers in water containing various acidic modifiers were applied to the resolution of valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved quite well on the two CSPs with the separation factors (α) in the range of 2.49～6.35 and resolutions (R_S) in the range of 2.95 ～ 12.21. Between the two CSPs, the CSP containing residual silanol protecting n-octyl groups on the silica surface was found to be better than the CSP containing residual silanol groups.

Full text of DOI:10.1002/chir.22424

CHIRALITY (2015)
Liquid Chromatographic Resolution of Amino Acid Esters of Acyclovir
Including Racemic Valacyclovir on Crown Ether-Based Chiral
Stationary Phases
*
SEONG AE AHN, AND MYUNG HO HYUN
Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan, Republic of Korea
ABSTRACT
Valacyclovir, a potential prodrug for the treatment of patients with herpes sim-
plex and herpes zoster, and its analogs were resolved on two chiral stationary phases (CSPs)
based on (3,3’-diphenyl-1,1’-binaphthyl)-20-crown-6 covalently bonded to silica gel. In order to
find out an appropriate mobile phase condition, various mobile phases consisting of various or-
ganic modifiers in water containing various acidic modifiers were applied to the resolution of
valacyclovir and its analogs. When 30% acetonitrile in water containing any of 0.05 M, 0.10 M,
or 0.15 M perchloric acid was used as a mobile phase, valacyclovir and its analogs were resolved
quite well on the two CSPs with the separation factors (α) in the range of 2.49 ~ 6.35 and resolu-
tions (RS) in the range of 2.95 ~ 12.21. Between the two CSPs, the CSP containing residual silanol
protecting n-octyl groups on the silica surface was found to be better than the CSP containing
residual silanol groups. Chirality 00:000–000, 2015. © 2015 Wiley Periodicals, Inc.
KEY WORDS: chiral stationary phase; enantiomer separation; liquid chromatography;
valacyclovir
Acyclovir (Fig. 1) is a highly effective antiviral drug for the
treatment of patients with herpes simplex and herpes zoster.1
However, the bioavailability of the drug at appropriate oral
doses is limited to about 20%. Consequently, multiple higher
oral doses or intravenous doses is necessary to reach suffi-
cient clinical results.1 The low oral bioavailability of acyclovir
might be due to weak intestinal absorption of the drug. To im-
prove the bioavailability of acyclovir, various amino acid es-
ters of acyclovir were developed as potential prodrugs.2
After oral administration, amino acid esters of acyclovir have
been known to be well absorbed from the intestinal tract
and then rapidly and extensively hydrolyzed to acyclovir, in-
creasing plasma levels of acyclovir. In particular, L-valyl ester
of acyclovir, valacyclovir (1) (Fig. 1), was found to show an
oral bioavailability of 3–5 times greater than that of acyclovir
itself.3,4 Interestingly, intestinal absorption of amino acid es-
ters of acyclovir was found to be stereoselective, with a prefer-
ence for L-isomers.2,5 In this instance, determination of the
enantiomeric composition of amino acid esters of acyclovir
is important.
For the determination of enantiomeric composition of chi-
ral drugs, liquid chromatographic enantioselective resolution
method with chiral stationary phases (CSPs) has been known
to be very effective.6 While the resolution of amino acid esters
of acyclovir on liquid chromatographic CSPs is quite rare,
Chiralpak AD column has been used to separate the two en-
antiomers of racemic valacyclovir3 and Crownpak CR (+) col-
umn has been used to identify L-valacyclovir in the presence
of some impurities.7
alcohols,9,11 fluoroquinolone antibacterials,12,13 racemic
catinone (central nervous system stimulant),14,15 and
tocainide (antiarrhythmic agent)16,17 were resolved very well
on CSP I and CSP II. In every case, CSP II was found to
show greater chiral recognition than CSP I. Amino acid
esters of acyclovir including racemic valacyclovir contain
one primary amino group. Consequently, they are expected
to be resolved on CSP I and CSP II. However, CSP I
and CSP II have not been applied to the resolution of ra-
cemic valacyclovir and other amino acid esters of acyclovir.
In this study, we report the liquid chromatographic resolu-
tion of various amino acid esters of acyclovir on CSP I and
CSP II.
EXPERIMENTAL
A high-performance liquid chromatography (HPLC) system consisting
of a Waters model 515 HPLC pump (Milford, MA), a Rheodyne model
7725i injector (Rohnert Park, CA) with a 20 μL sample loop, a Waters
2487 dual absorbance detector, and a YoungLin Autochro data module
(Software: YoungLin Autochro-WIN 2.0 plus) was used for the liquid
chromatography. The chiral column temperature was maintained at 20 °C
by using a JEIO TECH VTRC-620 cooling circulator (Daejeon, Korea).
Chiral columns packed with CSP I and CSP II were available from prior
studies.8,9 Injection samples were prepared by dissolving each analyte in
methanol at a concentration of 1.0 mg/mL and an injection size was typi-
cally 2.0 μL.
Racemic valacyclovir (1) and its analogs (2–5), shown in Figure 1,
were prepared via a procedure (Fig. 3) modified slightly from the proce-
dure reported for the preparation of L-valacyclovir.18 As an example, the
detailed synthetic procedure for the preparation of racemic valacyclovir
is described as follows. Racemic N-t-Boc-valine (0.25 g, 1.15 mmole),
which was prepared by treating racemic valine with di-tert-butyl
Previously, we developed two CSPs based on (3,3’-
diphenyl-1,1’-binaphthyl)-20-crown-6 (CSP I containing resid-
ual silanol groups on the silica surface and CSP II containing
residual silanol protecting n-octyl groups on the silica sur-
face, Fig. 2) covalently bonded to silica gel.8,9 The two CSPs
have been applied to the resolution of racemic compounds
containing a primary amino group. For example, α-amino
acids,8,9 β-amino acids,10 primary amines and amino
*Correspondence to: Myung Ho Hyun, Department of Chemistry, Pusan
National University, Busan 609-735, Republic of Korea.
E-mail: mhhyun@pusan.ac.kr
Received for publication 21 November 2014; Accepted 9 December 2014
DOI: 10.1002/chir.22424
Published online in Wiley Online Library
(wileyonlinelibrary.com).
© 2015 Wiley Periodicals, Inc..

AHN AND HYUN
Fig. 1. Structures of acyclovir, valacyclovir (1) and its analogs 2–5.
mmole), 4-(dimethylamino)pyridine (DMAP, 0.01 g, 0.082 mmole), and
N,N,N’,N’-tetramethylethylenediamine (TMEDA, 0.70 mL, 4.67 mmole).
The reaction mixture was stirred at 5 °C for 20 min and then O,O’-diethyl
chlorothiophosphate (DECTP, 0.30 mL, 1.91 mmole) was added drop by
drop. The reaction mixture was stirred at 5 °C for 2 h and then at room
temperature for 12 h. Water (100 mL) was added to the reaction mixture.
The reaction mixture was heated to 90 °C and then cooled to 5 °C slowly
with stirring. The precipitates were collected and washed with water to af-
ford N-t-Boc-valacyclovir (0.19 g, 66.9% yield). 1H NMR (CD3OD, ppm) δ
7.85 (s, 1H), 5.48 (s, 1H), 4.25-4.19 (m, 2H), 3.99-3.97 (d, 1H), 3.81-3.77
(m, 2H), 2.02-1.98 (m, 1H), 1.43 (s, 9H), 0.91-0.86 (m, 3H). N-t-Boc-
valacyclovir (0.05 g, 0.12 mmole) was dissolved in methylene chloride
(2 mL) in a 30-mL round-bottom flask. To the solution was added
trifluoroacetic acid (0.5 mL) and the whole mixture was stirred at room
temperature for 1 h. The reaction mixture was evaporated by using a ro-
tary evaporator to afford to racemic valacyclovir. 1H NMR (CD3OD,
ppm) δ 8.09 (s, 1H), 5.56 (s, 2H), 4.41-4.45 (m, 3H), 4.28-4.34 (m, 2H),
2.12-2.18 (m, 1H), 0.99 (d, 6H).
RESULTS AND DISCUSSION
Fig. 2. Structures of CSP I and CSP II.
Racemic valacyclovir (1) and its analogs (2–5) were re-
solved on CSP I and CSP II. As an effort to find out the most
widely applicable mobile phase condition, the five analytes
were resolved with variation of the type and content of organic
modifier in water containing perchloric acid (0.10 M) and the
resolution results are summarized in Table 1. In every case,
the L-enantiomer was found to be eluted faster than the D-
enantiomer. As an organic modifier, methanol (MeOH), etha-
nol (EtOH), or acetonitrile (ACN) was tested. Among the
three organic modifiers, acetonitrile was found to show the
best resolution results. As shown in Table 1, 30% acetonitrile
in water containing 0.10 M perchloric acid was found to be bet-
ter than 30% ethanol or 30% ethanol in water containing 0.10 M
perchloric acid as a mobile phase in terms of both the separa-
tion factors (α) and the resolutions (RS) for the resolution of
analytes 1–5 on CSP I and CSP II. When the content of aceto-
nitrile in water was changed from 15% to 30% and then to 50%,
the retention factors (k1) for the first eluted enantiomers were
found to decrease continuously in every case on both CSPs.
As an example, the chromatograms for the resolution of
Fig. 3. Scheme for the preparation of valacyclovir (1) and its analogs 2–5.
dicarbonate in the presence of triethylamine in a mixed solvent of dioxane
and water, was dissolved in dimethyl formamide (DMF, 20 mL) in a 250-
mL round-bottom flask. To the solution were added acyclovir (0.15 g, 0.67
Chirality DOI 10.1002/chir

RESOLUTION OF AMINO ACID ESTERS OF ACYCLOVIR
TABLE 1. Resolution of racemic valacyclovir (1) and its analogs (2–5) on CSP I and CSP II with variation of the content of methanol
(MeOH), ethanol (EtOH), or acetonitrile (ACN) in water as a mobile phase containing 0.10 M perchloric acid (HClO4)a
Organic
modifier in
1
2
3
4
5
CSP
water
k1
α
RS
k1
α
RS
k1
α
RS
k1
α
RS
k1
α
RS
CSP I
CSP I
CSP I
CSP I
CSP I
CSP II
CSP II
CSP II
CSP II
CSP II
MeOH (30%)
EtOH (30%)
ACN (30%)
ACN (15%)
ACN (50%)
MeOH (30%)
EtOH (30%)
ACN (30%)
ACN (15%)
ACN (50%)
1.30
0.68
0.87
1.43
0.35
0.60
0.22
0.45
0.81
0.12
2.43
2.45
2.88
2.61
3.16
2.34
2.63
2.77
2.62
3.80
2.50
2.04
3.22
3.71
2.65
2.65
2.52
4.43
5.41
2.63
3.06
1.57
2.08
3.22
1.14
1.13
0.41
0.87
1.47
0.36
2.85
2.86
2.97
2.85
3.15
2.97
3.30
3.18
3.11
3.84
3.96
3.50
4.82
4.69
4.33
4.59
4.25
8.00
8.32
6.40
6.38
2.88
3.94
7.43
1.15
3.02
1.03
1.86
4.09
0.42
2.85
2.85
3.46
3.10
3.69
2.99
2.94
3.62
3.35
4.47
3.62
3.39
5.04
5.06
4.84
3.54
4.20
6.51
6.10
7.00
5.97
3.13
3.83
6.77
1.61
2.14
0.89
1.67
3.17
0.53
5.04
5.04
5.27
5.08
5.36
5.52
5.89
6.01
5.83
7.06
4.29
5.13
5.43
6.75
6.36
4.28
7.17
8.90
9.24
11.42
4.10
2.16
3.13
5.03
1.20
1.77
0.73
1.41
2.80
0.38
4.90
4.79
5.47
5.11
5.64
5.11
5.30
6.10
5.80
7.64
4.71
4.53
6.13
6.50
6.23
4.26
6.06
8.56
8.51
9.55
aFlow rate: 0.5 ml/min. Detection: 254 nm UV. Column temperature: 20 °C. k1: Retention factor of the first eluted enantiomer. α: Separation factor. RS: Resolution.
valacyclovir (1) on CSP I and for the resolution of analyte 5 on
CSP II with the variation of the content of acetonitrile in water
containing 0.10 M perchloric acid are presented in Figure 4. In
general, the retention behaviors for the resolution of racemic
primary amino compounds on crown ether-based CSPs have
been explained by the balance between the hydrophilic inter-
action of the analytes with the mobile phase and the lipophilic
interaction of the analytes with the CSP.19,20 In reverse phase
chromatography, the lipophilic interaction of analytes with the
CSP might be an important factor for the retention of analytes,
especially when the CSP is lipophilic. Both CSP I and CSP II
might be lipophilic because of the 3,3’-diphenyl-1,1’-binaphthyl
group and CSP II is expected to be even more lipophilic be-
cause of the additional n-octyl groups.15 As the content of ace-
tonitrile in water is increased, the polarity of mobile phase
should decrease. In this instance, the lipophilic interaction be-
tween the analytes and the CSP is expected to decrease and,
consequently, the retention of analytes decreases as the con-
tent of acetonitrile in water is increased. The decreasing
trends of the retention factors are expected to be more signif-
icant with the more lipophilic CSP. Actually, the decreasing
trends of the retention factors are more significant with CSP
II than with CSP I, as expected. For example, the retention
factors for the resolution of valacyclovir (1) on CSP I and
CSP II were decreased by 76% and 85%, respectively, when
the acetonitrile content was changed from 15% to 50%, indicat-
ing that the decreasing trends of the retention factors are
more significant on the latter CSP. In general, the retention
times on CSP II are expected to be greater than those on
CSP I because of the higher lipophilicity of CSP II. However,
the retention factors (k1) on CSP II were smaller than those on
CSP I, as shown in Table 1. The dipolar interactions between
+
the primary ammonium ions (R-NH3 ) of analytes and the re-
sidual silanol groups on the silica surface of CSP I might be
more effective in retaining the analytes than the improved lipo-
philic interactions between the analytes and CSP II. In this in-
stance, the retention factors (k1) on CSP II could be smaller
than those on CSP I, but the exact reason is not yet clear.
When the content of acetonitrile in water was changed from
15% to 30% and then to 50%, the separation factors (α) were
found to increase in every case for the resolution of analytes
1–5 on CSP I and CSP II. As the content of acetonitrile in wa-
ter is increased, the retention times decrease for both of the
two enantiomers, for example, as shown in Figure 4. Retention
of two enantiomers on the CSPs is expected to be controlled
by the nonenantioselective and enantioselective interactions.
The nonenantioselective interactions such as dipolar interac-
tions and lipophilic interactions might be exerted equally on
the two enantiomers, but the enantioselective interactions
should be exerted more effectively on the more retained enan-
tiomer than the less retained enantiomer. As the content of
acetonitrile in water is increased, the nonenantioselective in-
teractions such as lipophilic interactions between the analytes
and the CSPs should decrease and the retention times of the
two enantiomers decrease, but the decreasing trends are less
significant with the more retained enantiomers because of the
more significant enantioselective interactions. As an example,
the retention factor of the less retained enantiomer for the
Fig. 4. Comparison of the chromatograms for the resolution of (a) valacyclovir (1) on CSP I and (b) analyte 5 on CSP II with the use of 15%, 30%, or 50% ace-
tonitrile (ACN) in water containing perchloric acid (0.10 M) as a mobile phase. Flow rate: 0.5 ml/min. Detection: 254 nm UV. Temperature: 20 °C.
Chirality DOI 10.1002/chir

AHN AND HYUN
resolution of valacyclovir (1) on CSP II decreases by 85% (re-
small enough to be useful and the separation factors and res-
olutions were quite good. Between the two acidic modifiers,
perchloric acid was found to be always better than
trifluoroacetic acid as an acidic modifier in terms of both the
separation factors and resolutions. As shown in Table 2, the
separation factors and resolutions for the five analytes are al-
ways greater with the use of perchloric acid (0.1 M) than with
the use of trifluoroacetic acid (0.1 M) as an acidic modifier on
both CSP I and CSP II. These results indicate that perchloric
acid is the best acidic modifier in 30% acetonitrile in water as a
mobile phase.
tention factor of the less retained enantiomer decreases from
0.81 to 0.12; see Table 1) while the retention factor of the more
retained enantiomer decreases by 78% (retention factor of the
more retained enantiomer decreases from 2.12 to 0.46; these
values are calculated from the data shown in Table 1) when
the content of acetonitrile in water is changed from 15% to
50%. Consequently, the separation factor (α) should increase
as the content of acetonitrile in water is increased on CSP I
and CSP II. In contrast, the resolutions (RS) for the resolution
of analytes 1–5 on CSP I and CSP II were found not to show
any consistent trend with the variation of the content of aceto-
nitrile in water.
The pH values of the mobile phases consisting of 30% ace-
tonitrile in water containing 0.10 M sulfuric, acetic,
trifluoroacetic, or perchloric acid measured at 20 °C were
0.89, 2.98, 1.05, and 0.99, respectively. The retention times
of analytes seem to be dependent on the mobile phase pH
values. When the pH value of the mobile phase is quite low
(0.89 for 0.10 M sulfuric acid), acid anion concentration is ex-
pected to be relatively high and, consequently, the ionic
strength of the mobile phase increases. In this instance, pri-
As an acidic modifier in mobile phase, four different acids
such as sulfuric acid, trifluoroacetic acid, acetic acid, and
perchloric acid were tested. The acidic modifier added to
the mobile phase has been proposed to protonate the primary
amino group of analytes and then the resulting primary am-
+
monium ion (R-NH3 ) of analytes can form energetically dif-
ferent two transient diastereomeric complexes inside the
cavity of the crown ether ring of the crown ether-based
CSPs.19,20 In this instance, acidic modifier in the mobile
phase is essential for the chiral recognition. The chromato-
graphic results for the resolution of five analytes on CSP I
and CSP II with the variation of the type and content of acidic
modifier in 30% acetonitrile in water as a mobile phase are
summarized in Table 2. When sulfuric acid was used as an
acidic modifier, the retention factors (k1) for the resolution
of five analytes on CSP I and CSP II were quite small. In
some cases (analyte 1 on CSP I, analytes 1 and 2 on CSP
II), the retention factors were too small to be useful when
sulfuric acid was used as an acidic modifier. In contrast,
the retention factors (k1) for the resolution of five analytes
1–5 on CSP I and CSP II were quite large when acetic acid
was used as an acidic modifier. The large retention factors
need long analytical times and a large volume of mobile
phase. Considering these results, it is concluded that sulfuric
acid or acetic acid is not appropriate as an acidic modifier for
the resolution of valacyclovir and its analogs on CSP I and
CSP II.
+
mary ammonium ions (R-NH3 ) of analytes are expected to
be hydrated significantly by the aqueous mobile phase and
then the protonated analytes could be distributed to the mo-
bile phase more significantly than to the stationary phase.20
Then the retention times of ionic analytes should be relatively
short. However, when the pH value of the mobile phase is
high (2.98 for 0.10 M acetic acid), the ionic strength of the
mobile phase is relatively low and, consequently, the reten-
tion times of ionic analytes should be relatively long. The
pH values of the mobile phase containing 0.10 M perchloric
acid is slightly lower than that of the mobile phase containing
0.10 M trifluoroacetic acid, but the retention times are longer
with the former mobile phase than with the latter mobile
-
phase. The lipophilicity of acid anion, ClO4 , is known to be
greater than that of CF3COO-.21 Consequently, primary am-
+
monium ions (R-NH3 ) of analytes containing counter acid an-
-
ion, ClO4 , are expected to be retained longer than those
containing CF3COO-. However, the trends of the separation
factors and resolutions with the variation of the type of acidic
modifier in aqueous mobile phase are not clear.
When trifluoroacetic acid or perchloric acid was used as an
acidic modifier in mobile phase, the retention factors were
When the concentration of perchloric acid was increased
from 0.05 M to 0.10 M, the retention factors (k1) increased
TABLE 2. Resolution of racemic valacyclovir (1) and its analogs (2–5) on CSP I and CSP II with variation of the content of acidic modifier
such as sulfuric acid (H2SO4), trifluoroacetic acid (TFA), acetic acid (AcOH), or perchloric acid (HClO4) in 30% acetonitrile (ACN) in
water as a mobile phasea
Acidic modifier
in mobile phase
1
2
3
4
5
CSP
k1
α
RS
k1
α
RS
k1
α
RS
k1
α
RS
k1
α
RS
CSP I
CSP I
CSP I
CSP I
CSP I
CSP I
CSP II
CSP II
CSP II
CSP II
CSP II
CSP II
H2SO4 (0.10 M)
TFA (0.10 M)
0.16
0.67
16.0
2.08
1.44
1.78
2.74
2.84
2.49
2.97
2.87
3.02
1.17
3.26
3.93
4.82
4.40
4.63
0.31
1.26
29.4
3.94
2.43
2.86
0.19
0.77
13.7
1.86
1.21
1.29
3.69
3.36
3.05
3.46
3.40
3.58
3.45
3.26
2.83
3.62
3.45
3.79
2.77
4.36
4.61
5.04
5.22
5.88
3.31
6.34
2.81
6.51
6.42
8.17
0.32
1.39
24.8
3.83
2.82
3.32
0.20
0.70
14.7
1.67
1.16
1.27
5.82
5.11
4.76
5.27
5.07
5.31
4.85
5.63
4.38
6.01
5.66
6.22
4.14
5.41
4.60
5.43
6.81
7.24
4.56
9.79
5.48
8.90
10.82
12.21
0.25
1.08
25.7
3.13
2.12
2.44
0.12
0.60
11.5
1.41
0.96
1.02
6.14
5.31
4.78
5.47
5.26
5.56
5.61
5.52
4.56
6.10
5.70
6.35
3.95
5.68
5.59
6.13
6.44
6.91
4.60
8.29
3.21
8.56
9.26
10.08
0.29
10.4
0.87
0.61
0.68
2.79
2.05
2.88
2.73
3.02
1.95
3.42
3.22
2.95
3.32
AcOH (0.10 M)
HClO4 (0.10 M)
HClO4 (0.05 M)
HClO4 (0.15 M)
H2SO4 (0.10 M)
TFA (0.10 M)
AcOH (0.10 M)
HClO4 (0.10 M)
HClO4 (0.05 M)
HClO4 (0.15 M)
0.21
6.79
0.45
0.35
0.31
2.42
1.64
2.77
2.49
3.04
2.56
2.00
4.43
3.70
4.80
0.36
10.4
0.87
0.64
0.69
2.75
2.27
3.18
3.00
3.33
4.38
5.80
8.00
6.84
7.79
aFlow rate: 0.5 ml/min. Detection: 254 nm UV. Column temperature: 20 °C. k1: Retention factor of the first eluted enantiomer. α: Separation factor. RS: Resolution.
Chirality DOI 10.1002/chir

RESOLUTION OF AMINO ACID ESTERS OF ACYCLOVIR
Fig. 5. Chromatograms for the resolution of the mixture of analytes 1–5 on (a) CSP I and (b) CSP II with the use of 30% acetonitrile in water containing
perchloric acid (0.10 M) as a mobile phase. Flow rate: 0.5 ml/min. Detection: 254 nm UV. Temperature: 20 °C.
in every case. However, when the concentration of perchloric
acid was increased further from 0.10M to 0.15M, the retention
factors (k1) decreased. In contrast, the separation factors (α) for
the resolution of five analytes on CSP I and CSP II were found
to increase steadily as the concentration of perchloric acid in
aqueous mobile phase is increased. The resolutions (RS) were
found not to show consistent trends with the variation of
perchloric acid concentration in aqueous mobile phase, but
analytes 1, 3, 4, and 5 show the highest resolutions on CSP I
and CSP II when the concentration of perchloric acid is
0.15M. Between the two CSPs, CSP II was found to be better
than CSP I except for the resolution of valacyclovir (1) in terms
of the separation factors (α) and CSP II was found to be always
better than CSP I in terms of the resolutions (RS) when 30% ace-
tonitrile in water containing perchloric acid was used as a mo-
bile phase. The protection of the residual silanol groups on
the silica surface of CSP I with n-octyl groups in CSP II is
expected to remove the nonenantioselective interaction sites
and consequently improve the chiral recognition.9 Overall, the
separation factors are always greater than 2.49 and the resolu-
tions are greater than 2.95 on CSP I and CSP II with the use
of 30% acetonitrile in water containing perchloric acid. In this
instance, 30% acetonitrile in water containing any of 0.05M,
0.10M, or 0.15M perchloric acid is expected to be useful for
the resolution of valacyclovir and its analogs. Previously,
valacyclovir was reported to be resolved on a Chiralpak AD
column with the separation factor of 2.00 and a resolution of
4.10.3 In contrast, the separation factors for the resolution of
valacyclovir on CSP I and CSP II were 3.02 and 3.04, respec-
tively, when 30% acetonitrile in water containing 0.15M
perchloric acid was used as a mobile phase. The resolution
values for the resolution of valacyclovir on CSP I and CSP II
were 3.32 and 4.83, respectively, under the identical mobile
phase condition. From these results, both CSP I and CSP II
are concluded to be much better than a Chiralpak AD column
for the resolution valacyclovir in terms of separation factor.
CSP II is also concluded to be better than Chiralpak AD column
for the resolution of valacyclovir in terms of resolution.
In conclusion, two CSPs (CSP I and CSP II) based on (3,3’-
diphenyl-1,1’-binaphthyl)-20-crown-6 covalently bonded to sil-
ica gel were found to be quite successful for the resolution
of valacyclovir and its analogs. When 30% acetonitrile in water
containing any of 0.05 M, 0.10 M, or 0.15 M perchloric acid
was used as a mobile phase, the separation factors (α) were
in the range of 2.49 ~ 6.35 and the resolutions (RS) were in
the range of 2.95 ~ 12.21 on the two CSPs. From these results,
it is concluded that either of the two CSPs can be successfully
utilized for the determination of the enantiomeric composi-
tion of valacyclovir and its analogs with the use of 30% aceto-
nitrile in water containing any of 0.05 M, 0.10 M, or 0.15 M
perchloric acid as a mobile phase.
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