A kinetic oxymoron: Concentration-dependent first-order rate constants for hydrolysis of ceftazidime

Article abstract of DOI:10.1021/js970179k

The influence of pH, temperature, and buffers on the hydrolysis of 10^{- 4} M ceftazidine was previously reported. The pH-rate profiles showed that maximum stability occurred in the pH-independent region from 4.5 to 6.5. In the present study, hydrolysis rates of 0.031, 0.14, 0.25, and 0.35 M ceftazidime were measured at 30 and 65°C, pH 5.5-6.2. The data were consistent with β-lactam hydrolysis and the rapid release of pyridine. The sum of the time-dependent concentrations of ceftazidime and pyridine provided mass balance. Simultaneous nonlinear regression for ceftazidime loss and pyridine formation provided similar rate constants (k) to those determined from first-order plots of ceftazidime loss. Although the loss of ceftazidime was first-order for each initial concentration, the k values increased as the initial concentrations increased. Plots of k versus initial concentration were linear with intercepts similar to the k values for 10^-4 M solutions, thus implying that ceftazidime catalyzed its own degradation. At the pH of these studies ceftazidime exists as a base. The ceftazidime catalytic constant, calculated from the slope of the plot, was similar to that found for the general-base catalyst, HPO₄^2-. Therefore, it is feasible that ceftazidime also behaved as a intermolecular general-base catalyst. However, first-order plots exhibited excellent linearity even though the catalyst (ceftazidime) was consumed. This would require that the catalytic moieties on ceftazidime remained relatively constant throughout its hydrolysis. This hypothesis was shown to be consistent with literature reports which indicate that the general-base catalytic groups can remain relatively constant during cephalosporin hydrolysis.

Full text of DOI:10.1021/js970179k

A Kinetic Oxymoron: Concentration-Dependent First-Order Rate
Constants for Hydrolysis of Ceftazidime
JOSEPHINE O. FUBARA AND ROBERT E. NOTARI*
Contribution from the The Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina,
71 Ashley Avenue Charleston, South Carolina 29425.
1
Received May 5, 1997.
FXinal revised manuscript received October 17, 1997.
Accepted for
publication October 20, 1997 .
Abstract
0 The influence of pH, temperature, and buffers on the
-4
hydrolysis of 10 M ceftazidime was previously reported. The pH
−
rate profiles showed that maximum stability occurred in the pH-
independent region from 4.5 to 6.5. In the present study, hydrolysis
rates of 0.031, 0.14, 0.25, and 0.35 M ceftazidime were measured at
30 and 65 °C, pH 5.5−6.2. The data were consistent with â-lactam
hydrolysis and the rapid release of pyridine. The sum of the time-
dependent concentrations of ceftazidime and pyridine provided mass
balance. Simultaneous nonlinear regression for ceftazidime loss and
pyridine formation provided similar rate constants (k) to those
determined from first-order plots of ceftazidime loss. Although the
loss of ceftazidime was first-order for each initial concentration, the k
values increased as the initial concentrations increased. Plots of k
versus initial concentration were linear with intercepts similar to the k
-4
values for 10 M solutions, thus implying that ceftazidime catalyzed
its own degradation. At the pH of these studies ceftazidime exists
as a base. The ceftazidime catalytic constant, calculated from the
slope of the plot, was similar to that found for the general-base catalyst,
2-
HPO
4
. Therefore, it is feasible that ceftazidime also behaved as a
intermolecular general-base catalyst. However, first-order plots
exhibited excellent linearity even though the catalyst (ceftazidime) was
consumed. This would require that the catalytic moieties on
ceftazidime remained relatively constant throughout its hydrolysis. This
hypothesis was shown to be consistent with literature reports which
indicate that the general-base catalytic groups can remain relatively
constant during cephalosporin hydrolysis.
Scheme 1
1
0
and pH values between ∼5 and ∼8.
Thus, clinical
solutions of ceftazidime have all of the prerequisites for
dimerization, i.e., high concentrations, alkaline pH, an
amine, and a â-lactam. Although ceftazidime dimerization
has not been reported, its potential for dimerization at
clinically employed concentrations prompted the current
investigation.
The kinetics of dimerization of several aminopenicillins
1
-3
have been reported by Bundgaard.
Dimerization oc-
curred via intermolecular nucleophilic attack by a side
chain primary amine of one molecule on the â-lactam
carbonyl carbon of a second. Storage of slightly alkaline,
concentrated solutions of â-lactam antibiotics with side
Previous research in this laboratory has defined the
influence of pH, temperature and buffers on the first-order
hydrolysis of ceftazidime in dilute aqueous solutions.
Hydrolysis rate constants were pH-independent in the
region 4.5-6.5. The goal of this research was to define the
kinetics of ceftazidime degradation in the pH-independent
region with concentrations within the clinically employed
range.
chain amines has resulted in further reaction to form
11
polymers.4
-7
These polymers have been found to be anti-
genic and capable of inducing cellular immunity in ani-
mals.5
,6,8
Ceftazidime is a semisynthetic, broad-spectrum cepha-
losporin with a primary amine on a thiazole ring (Scheme
1
). While numerous dimerization and polymerization
studies have been conducted with penicillins having pri-
mary amines, relatively few have been reported for ami-
nocephalosporins. A dimer has been isolated from 50% w/v,
pH 8.5, aqueous solutions of cefotaxime which has struc-
Experimental Section
Ma ter ia lssCeftazidime pentahydrate was used as provided by
SmithKline Beecham Pharmaceuticals (Philadelphia). All other
chemicals were analytical or HPLC grade.
tural similarities to ceftazidime that include an aminothia-
zole (Scheme 1).9
Ceftazidime is clinically employed in aqueous solutions
Cefta zid im e HP LC Assa yssThe system consisted of a Waters
M-510 solvent delivery module, a Waters M-481 variable wave-
length detector, a Shimadzu C-R3A integrator, and a Rheodyne
M-7125 manual injector. The injection volume was 20 µL.
Chromatographic separations (Figure 1) were achieved on a
ranging from ∼10 to ∼280 mg/mL (∼0.02 M to ∼0.5 M)
*
Phone (803) 792-8426. Fax (803) 792-0759. E-mail: notariro@
MUSC.EDU.
X
Abstract published in Advance ACS Abstracts, December 15, 1997.
©
1998, American Chemical Society and
S0022-3549(97)00179-2 CCC: $15.00
Published on Web 01/02/1998
Journal of Pharmaceutical Sciences / 53
American Pharmaceutical Association
Vol. 87, No. 1, January 1998

Figure 2
s
Typical first-order plots for loss of 0.35, 0.14, and 0.031 M
ceftazidime in unbuffered aqueous solutions at 65
°
C, pH 5.5 6.2.
−
In addition to the HPLC evidence, reactions in which 50 to 80%
of the initial ceftazidime had degraded were freeze-dried. Part of
the residue was dissolved in acetone and analyzed by GC/MS. The
resulting mass spectrum matched that obtained with a pure
sample of pyridine. The remaining residue was dissolved in D2O,
analyzed with proton nuclear magnetic resonance (NMR), and
found to have peaks that were also obtained using pure pyridine.
Cefta zid im e Hyd r olysis Kin eticssThe pH values for the
reaction solutions were measured before and after each reaction
at the experimental temperatures, 30 and 65 °C (stable to (0.1
°C). The first-order hydrolysis rate constants of ceftazidime were
previously shown to be pH-independent in the pH region of ∼4.5
Figure 1
s
HPLC chromatograms as a function of time during hydrolysis of
.35 M ceftazidime at 30 C, pH 5.5 6.2. The retention time for ceftazidime
5.3 min (1) and that for pyridine is 3.3 min (2). The other peaks were
0
°
−
is
∼
∼
not identified.
Spherisorb C-6 column (5µm, 4.6-mm i.d. x 150 mm; Keystone
Scientific Inc., State College, PA) at ambient temperature with a
flow rate of 1.4 mL/min and UV detection at 258 nm. Mobile
phases were filtered through a type HA, 0.45-µm membrane filter
(Millipore Corporation) and deaerated under reduced pressure.
The quantitative separation of ceftazidime from its degradation
1
1
products was achieved with a mobile phase consisting of aqueous
.10 M acetic acid and 0.05 M sodium acetate with 6% (v/v)
to ∼6.5. Aqueous solutions containing empirically determined
concentrations of sodium hydroxide were prepared to facilitate
dissolution and provide initial pH values of ∼5.5 at the reaction
temperatures for all initial ceftazidime concentrations. Following
the reactions, the final pH values were determined to be 5.8-6.2.
Reactions were initiated by dissolving sufficient ceftazidime in
10 mL of the appropriate sodium hydroxide solution at 30 or 65
°C to provide initial concentrations of 0.031, 0.14, 0.25, or 0.35 M.
A sample was removed as a function of time and cooled, and a 35
µL aliquot was diluted with 10 mL of water. Most dilutions were
assayed within 12 h after sampling. No loss of ceftazidime was
detected by HPLC when these dilutions were stored for 24 h in
the refrigerator.
Buffers were not employed for three reasons. It was possible
to maintain the pH within the pH-independent region without the
addition of buffers. Previous studies have shown that ceftazidime
hydrolysis is catalyzed by buffers.11 The ceftazidime solutions
were so concentrated that they would require high concentrations
of buffers relative to the substrate concentrations to represent
significant buffer capacity.
0
acetonitrile. The ceftazidime retention time was 5.3 min. Four
linear calibration plots were constructed to accommodate the four
initial concentrations. Reactions were diluted for analysis into
-
4
-3
-4
the ranges (in M) 1.8 × 10 to 1.6 × 10 , 1.3 × 10 to 1.2 ×
-
3
-5
-4
-5
-4
1
0
, 7.0 × 10 to 6.3 × 10 , and 1.5 × 10 to 1.4 × 10 . The
coefficient of variation (CV) for ceftazidime analysis varied from
.2 for the highest concentration range to 2 for the lowest
1
concentration range. Recovery was 98 ( 3%.
Several methods were employed to ensure that assays were
specific for ceftazidime in the presence of its degradation products.
No residual peaks were found beneath the ceftazidime peak when
reactions were allowed to proceed to completion. No deviations
from linearity were observed in the first-order plots. Representa-
tive reactions were analyzed in duplicate with the mobile phase
described above and a second mobile phase that provided a
ceftazidime retention time of 7.2 min. This mobile phase consisted
of aqueous 0.10 M acetic acid and 0.05 M sodium acetate with 4%
(v/v) acetonitrile. The concentrations and first-order plots obtained
for both methods were similar. Furthermore, as discussed in the
Results, the first-order rate constants based on the formation of a
degradation product (pyridine) agreed with those determined from
ceftazidime loss.
Results
An a lysis of P yr id in e in Rea ction Solu tion ssThe reaction
solutions were found to acquire a characteristic pyridine odor as
ceftazidime hydrolyzed. Pyridinium was found to be the best
leaving group in a study which compared hydrolysis rates of 33
cephalosporins with various 3-methylene substituents.12 There-
fore, ceftazidime degradation was expected to release pyridine just
as the hydrolysis of chemically similar cefpirome (Scheme 1)
Deter m in a tion of th e Kin etic Or d er for Cefta zi-
d im e LosssFor a given starting concentration at constant
temperature and pH, ceftazidime loss was described by
first-order kinetics. Pseudo-first-order rate constants (k)
14
were calculated from first-order plots based on eq 1
1
3
ln [C] ) ln [C ] - kt
(1)
released 2,3-cylopentenopyridine.
Solutions were prepared to
0
represent ceftazidime:pyridine ratios of 9:1, 5:5, and 2:8 and
assayed by HPLC. The retention times for the ceftazidime and
pyridine HPLC peaks in these mixtures agreed with those from
the reactions (Figure 1). The molar absorptivity for ceftazidime
at 258 nm is ∼7 times greater than that for pyridine. Therefore,
samples were more concentrated to assay pyridine relative to those
for the ceftazidime assays. Reaction aliquots of 50 µL were added
to 5 mL of water and assayed for pyridine. Linear calibration plots
were constructed to assay these dilutions over the concentration
where the initial concentration of ceftazidime is [C ], the
time-dependent concentration is [C], and t is time. Each
study was comprised of eight or more assays spaced to
0
provide changes of ∼0.1[C
were linear over the decrease in concentration from [C
] (Figure 2). Rate constants were obtained by
0
] per sampling interval. Plots
0
]
to ∼0.2[C
0
2
linear regression. For replicate studies the r values
exceeded 0.99, CV < 5%, and for duplicate studies the
differences were <4% (Table 1).
-3
-3
range 0.43 × 10 to 4.72 × 10 M. The CV was 1.5 and recovery
was 98 ( 1.5%.
5
4 / Journal of Pharmaceutical Sciences
Vol. 87, No. 1, January 1998

Table 1
sExperimental Conditions and Observed First-Order Rate
2 1 a
-
Constants (10 k in h ) for Hydrolysis of Ceftazidime
2
-
r2
10 k (h 1)c
2
-
r2
°
C
concn (M)
0.35
10 k (h 1)b
6
5
20.0
0.5
9.3
0.4
0.999
0.999
0.999
0.998
s
s
s
s
s
s
s
s
2
1
2
mean (CV)
.25
20.0(
±
±
±
2.7%)
4.3%)
2.4%)
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
0
18.5
0.999
0.998
0.998
1
1
7.3
7.1
mean (CV)
.14
17.6(
Figure 3
s
Typical second-order plots for loss of 0.35 and 0.14 M ceftazidime
in unbuffered aqueous solutions at 65
°
C, pH 5.5 6.2. The solid lines are
−
0
14.9
0.997
0.997
0.998
based on eq 2 and the dashed lines connecting the data points show their
deviation from linearity.
1
1
4.2
4.5
mean (CV)
.031
14.5(
0
10.3
0.999
0.999
0.998
9
9
.60
.48
mean (CV)
0.35
9.79(
0.720
±
4.5%)
s
s
30
0.999
0.999
0.999
0.999
0.708
0.720
0.725
0.722
0.719(
0.618
0.618
0.618(
0.999
0.999
0.996
0.997
0.720
0.692
0.701
mean (CV)
0.708(
0.612
±
±
2.0%)
1.9%)
±
±
1.0%)
0.0%)
0
.25
mean (range)
.14
0.999
0.999
0.999
0.999
0.636
0.624(
Figure 4
C, pH 5.5
ceftazidime concentration: (A) ceftazidime (
the sum of ceftazidime and pyridine as a function of time (2
s
Mass balance data during the hydrolysis of 0.35 M ceftazidime at
6.2. Concentrations are expressed as percentage of the initial
); (B) pyridine ( ); and (C) is
). The curves
30
°
−
0
0.528
0.999
0.999
0.516
0.526
0.521(
s
s
s
0.999
0.999
b
9
0.516
mean (range)
.031
0.522(
0.378
±
±
1.1%)
0.8%)
±
1.1%)
were obtained by simultaneous nonlinear regression with eqs 3 and 4 where
[Cef ] and [Pyr ] were the percentage concentrations.
0
0.998
0.999
s
s
s
0.384
mean (range)
0.381(
neously determined as a function of time. Figure 4 shows
typical data for the time-dependent simultaneously mea-
sured concentrations of ceftazidime and pyridine. The sum
of these concentrations as a function of time was consistent
with the starting concentration of ceftazidime. Recovery
was 98% (( 3%) as a function of time.
a
Initial pH values were 5.5 and final pH values were between 5.8 and 6.2.
Under these conditions hydrolysis rate constants were reported to be pH-
independent.
with eq 1. Determined by simultaneous nonlinear regression of data for
ceftazidime loss and pyridine formation with eqs 3 and 4.
11
b
Determined from linear first-order plots for ceftazidime loss
c
Thus, mass balance indicated that the loss of ceftazidime
was accompanied by the formation of pyridine. These time-
dependent data were analyzed by simultaneous nonlinear
regression based on eqs 3 and 4
Deviation from first-order rates is usually associated
with higher order reactions such as the dimerization of
amino â-lactam antibiotics.1
-3
Ceftazidime has an unpro-
tonated amine at the pH values of this study. Therefore,
additional measures were taken to ensure that the kinetics
of ceftazidime loss did not indicate dimerization. Dimer-
ization is represented by a second-order reaction, C + C f
-
kt
[Cef] ) [Cef ]e
(3)
(4)
o
Pyr] ) [Cef ]{1 - e-kt}
[
o
2
C . Therefore, the data were also analyzed using the
1
4
second-order eq 2
where [Cef] and [Pyr] are the concentrations of ceftazidime
and pyridine as a function of time, t; [Cef
] is the initial
o
1
/[C] ) 1/[C ] + kt
(2)
0
ceftazidime concentration and k is the first-order rate
constant for ceftazidime loss and pyridine formation.
Simultaneous nonlinear regression for ceftazidime loss and
pyridine formation provided first-order hydrolysis rate
constant values that agreed with those determined from
first-order plots of ceftazidime loss (Table 1).
Second-order behavior would be most evident in the most
concentrated solutions. Figure 3 shows typical nonlinear
second-order plots for 0.35 and 0.14 M ceftazidime. The
inability of eq 2 to describe the data for any of the initial
concentrations demonstrated that ceftazidime loss was not
second-order. Furthermore, second-order data were syn-
thesized to represent each of the sampling intervals and
to provide 80% loss of ceftazidime over the experimental
time. First-order plots for these data were not linear.
However, no deviations from linearity were observed for
the first-order plots of the experimentally measured ceftazi-
dime loss.
Discussion
E vid en ce for F ir st -Or d er H yd r olysis of Ceft a zi-
d im esThe current studies were conducted at pH 5.5-6.2,
which is the pH-independent region in ceftazidime hydroly-
sis pH-rate profiles.11 Therefore, degradation would be
primarily due to spontaneous (non-pH-catalyzed) water
attack resulting in hydrolysis of the ceftazidime â-lactam.
All first-order plots for ceftazidime concentration as a
function of time showed excellent linearity (Figure 2).
Ma ss Ba la n ce a n d Kin etic An a lyses of Cefta zid im e
Loss a n d P yr id in e F or m a tion sPyridine was identified
as a degradation product by HPLC, GC/MS, and NMR. The
concentrations of ceftazidime and pyridine were simulta-
1
5
Journal of Pharmaceutical Sciences / 55
Vol. 87, No. 1, January 1998

As part of a previous study on the hydrolysis of ce-
furoxime (Scheme 1), a commercial product was reconsti-
tuted by dissolving 750 mg in 100 mL of water, in
1
9
accordance with the prescribing information.
The hy-
drolysis rate constant for this ∼0.02 M cefuroxime solution
was adequately predicted from first-order hydrolysis con-
stants and their energetics, which were determined from
-
4
kinetic studies of 10 M solutions at elevated tempera-
tures. However, cefuroxime hydrolysis was not catalyzed
by formate, acetate, phosphate, or borate buffers. In
contrast, previous research in this laboratory has demon-
strated that every one of these buffers catalyzed the
hydrolysis of ceftazidime.11 Thus, ceftazidime hydrolysis,
which is subject to buffer catalysis, elicits concentration-
dependent first-order rate constants, whereas cefuroxime
hydrolysis does not exhibit either of these traits. This
suggests that ceftazidime is catalyzing its own hydrolysis.
The ceftazidime carboxylic acids and the protonated
Figure 5sFirst-order hydrolysis rate constants (b) as a function of the initial
ceftazidime concentrations for reactions initiated at pH 5.5 with final pH values
2
0
-
4
a
aminothiazolyl have pK values of 1.9, 2.7, and 4.1.
e
6.2. The data at the intercept represent the hydrolysis of 10 M ceftazidime
11
at pH 5.5 (
9
) and 6.2 (
2
) in the absence of buffer catalysis at 65
dashed line, which shows the first-order rate constants as a function of HPO
°
C. The
Therefore ceftazidime would be in the unprotonated form
at the pH values of this study. These three basic groups
should be capable of acting as intermolecular general-base
catalysts on the basis of the previously observed catalysis
by the basic components in formate, acetate, phosphate,
2-
4
2-
concentration, was calculated with the HPO
4
catalytic constant of 0.349
) is the reported intercept of k versus phosphate
) was calculated with the pH-
-
1
-
1 11
M
h
.
The pH 5.5 value (9
buffer concentration and the pH 6.2 value (
rate expression.
2
11
11
and borate buffers.
As the ceftazidime concentration was increased from 10-
4
Furthermore, second-order plots that would accompany
dimerization were not linear (Figure 3).
to 0.35 M, the hydrolysis rate constant at 65 °C increased
-1
from 0.09 to 0.20 h (Figure 5). The value of the apparent
Additional evidence for first-order hydrolysis was ob-
tained from the kinetic analysis of pyridine formation.
Results of the simultaneous analysis of the concentration
of ceftazidime and pyridine as a function of time, which
include mass balance (Figure 4) and agreement of the
hydrolysis rate constants (Table 1), were consistent with
â-lactam hydrolysis followed by rapid release of pyridine.
The significance of this agreement in rate constants can
be realized by considering what is known about the
hydrolysis of cephalosporins. It is well-established that
hydrolysis of the â-lactam ring of cephalosporins, having
a suitable leaving group at the 3-methylene position,
results in rapid release of that group and formation of
catalytic constant, calculated from the slope of this line, is
-1
-1
0
.314 M
h
. The feasibility of this value representing
intermolecular general-base catalysis by ceftazidime can
be assessed by comparing it to a known general-base
catalyst.
The pH region of the current study corresponds to the
11
phosphate buffer region in our previous report.
The
dashed line in Figure 5 illustrates the dependency of the
first-order rate constants for ceftazidime hydrolysis on the
2-
concentration of HPO
4
at 65 °C according to eq 8:
k ) kpH + kHPO4[HPO42-]
(8)
1
2,13,16-18
unstable exomethylene compounds.
Thus the data
where kpH ) 0.0875 h- is the value in the absence of buffer
1
for the release of pyridine would be expected to mirror the
data for hydrolysis of ceftazidime.
Th e Kin etic Oxym or on a n d Its Ra tion a liza tion sA
true first-order rate process is described by eq 5:
-
1
-1
catalysis and kHPO4 ) 0.349 M
h
is the catalytic constant
2
-
at 65 °C.11 As shown in Figure 5, HPO
2-
for HPO
the capacity to increase the hydrolysis constant from 0.0875
4
4
has
-
1
2-
-1
h
in the absence of HPO
4
to 0.210 h in the presence
-
kt
of 0.35 M HPO
2-
. The dashed line is similar to that of
4
[
C] ) [C ]e
(5)
o
the ceftazidime line because the apparent catalytic constant
for ceftazidime is comparable to the catalytic constant for
Equation 5 can be rearranged and the logarithmic
transformation provides eq 6:
2-
HPO
4
. Although the quantitative similarity between
catalytic constants is fortuitous, their agreement supports
the hypothesis that it is feasible for ceftazidime to behave
as a intermolecular general-base catalyst.
ln([C]/[C ]) ) ln F ) -k t
(6)
o
Therefore, the apparent catalytic constant suggests that
ceftazidime loss was accelerated as its initial concentra-
tions were increased because of intermolecular general-
base catalysis by the aminothiazolyl and carboxylate
groups. However, first-order plots exhibited excellent
linearity even though the catalyst (ceftazidime) was con-
sumed. This observation requires that the total catalytic
activity must remain reasonably constant during the loss
of ceftazidime. Such would be the case if the catalytic
groups either remained intact throughout the reaction or
were replaced by equivalent catalysts in reaction products.
Is Th is Ra tion a liza tion in Accor d w ith Kn ow n
Cep h a losp or in Hyd r olysis P r od u cts?sThe proposed
explanation for this unusual phenomenon is consistent with
all known kinetic information. It must also be compatible
with known cephalosporin hydrolysis chemistry. The data
for ceftazidime loss and pyridine formation conform to a
where F ) ([C]/[C
o
]) or the fraction remaining at time, t.
Equation 6 may be written as eq 7
k ) -ln F/t
(7)
This clearly shows why a true first-order rate constant
is independent of the starting concentration since only the
fraction remaining appears in the equation.
In the current study, apparent first-order kinetics were
observed for each initial ceftazidime concentration. How-
ever, contrary to eq 7, the observed first-order rate constant
values increased as the initial concentrations increased
(Figure 5). It was therefore necessary to rationalize the
observation of concentration-dependent first-order rate
constants for solutions that contained only ceftazidime and
water.
5
6 / Journal of Pharmaceutical Sciences
Vol. 87, No. 1, January 1998

carboxylate originally located at the C-2 position. However,
the loss of this catalyst would be offset by the formation of
pyridine, which exists ∼80% in the unprotonated form at
pH ∼5.8. Overall, the side chain carboxylate and ami-
nothiazole groups would remain intact while loss of the C-2
carboxylate catalyst would be counterbalanced by pyridine
formation. Consequently, although the relative contribu-
tion of each ceftazidime general-base catalytic group is not
known, the total concentration of the potential general-
base catalysts would remain practically constant in Scheme
2
.
Sign ifica n cesSubstrate concentration-dependent first-
order rate constants have been observed for electrode
reactions involving complex ion radicals in nonaqeous
solvents where diffusion-limited kinetics have rate law
2
3
terms with substrate concentration in the denominator.
However, the current observation appears to be unprec-
edented for simple first-order hydrolysis in aqueous sys-
tems containing only substrate and water. A literature
search did not disclose any previously reported substrate-
concentration dependent first-order rate constants for such
simple systems.
While ceftazidime hydrolysis in concentrated solutions
appears to present a unique kinetic situation, the phenom-
enon may be of more widespread occurrence. Substrate-
concentration accelerated first-order hydrolysis is feasible
whenever hydrolysis is subject to general-acid and/or
general-base catalysis, the substrate has one or more
effective general-acid and/or general-base catalytic groups,
the total concentration of the catalytic groups remains
practically constant throughout the reaction, and the initial
concentration is sufficiently large to elicit measurable
intermolecular catalysis.
Scheme
2
s
Expected products for ceftazidime degradation at pH 5.8
based on
â-lactam hydrolysis pathways for cephalosporins with leaving
12,13,16-18
groups at the 3-methylene position.
widely recognized â-lactam hydrolysis pathway for cepha-
References and Notes
losporins with suitable leaving groups at the 3-methylene
position.1
2,13,16-18
For such cases, water cleaves the â-lac-
1
. Bundgaard, H. Polymerization of Penicillins: Kinetics and
Mechanism of Di- and Polymerization of Ampicillin in
Aqueous Solution. Acta Pharm. Suec. 1976, 13, 9-26.
2. Bundgaard, H. Polymerization of Penicillins: II Kinetics and
Mechanism of Dimerization and Self-Catalyzed Hydrolysis
of Amoxycillin in Aqueous Solution. Acta Pharm. Suec. 1977,
tam ring15 to form a cephalosporoate which spontaneously
releases the leaving group. This results in the formation
of an unstable exomethylene imine16 which undergoes
hydrolytic fission at the C
an intermediate aldehyde which decarboxylates.
6
-N
1
and C
6
5
-S bonds to form
1
3,17,18
This
1
4, 47-66.
pathway has been reported for cepirome, cefsulodin,17 and
many others.18 For example, at 40 °C, pH 7, the primary
degradation pathway for cefpirome, which is structurally
similar to ceftazidime (Scheme 1), involved hydrolysis of
the â-lactam followed by the loss of 2,3-cyclopentenopyri-
dine and formation of an unstable exomethylene interme-
13
3. Bundgaard, H. Polymerization of Penicillins: III Structural
Effects Influencing Rate of Dimerization of Amino-Penicillins
in Aqueous Solution. Acta Pharm. Suec. 1977, 14, 67-80.
. Bundgaard, H.; Larsen, C. Polymerization of Penicillins: IV
Separation, Isolation and Characterization of Ampicillin
Polymers formed in Aqueous Solution. J . Chromatogr. 1977,
132, 51-59.
. Dewdney, J . M.; Smith, H.; Wheeler, A. W. The Formation
of Antigenic Polymers in Aqueous Solutions of â-Lactam
Antibiotics. Immunology 1971, 21, 517-525.
. Smith, H.; Dewdney, J . M.; Wheeler, A. W. A Comparison of
the Amounts and the Antigenicity of Polymeric Materials
Formed in Aqueous Solution by Some â-Lactam Antibiotics.
Immunology 1971, 21, 527-533.
. Smith, H.; Marshall, A. C. Polymers Formed by Some
â-Lactam Antibiotics. Nature (London) 1971, 232, 45-46.
. Bundgaard, H. Penicillin Allergy: Kinetics of Penicilloylation
of Serum Albumins by Various Penicillins. Acta Pharm. Suec.
1977, 14, 391-402.
. Zhang, Q. Polymerization Properties of Cefotaxime. Chung-
Kuo Kang Sheng Su Tsa Chih. 1992, 17, 428-434.
0. Tazicef. In Physicians’ Desk Reference, 51st ed.; Medical
Economics Company Inc.: Montvale, NJ , 1997; pp 2697-
2701.
1. Zhou, M.; Notari, R. E. Influence of pH, Temperature and
Buffers on the Kinetics of Ceftazidime Degradation in
Aqueous Solutions. J . Pharm. Sci. 1995, 5, 534-538.
2. Coene, B.; Schanck, A.; Dereppe, J -M.; VanMeerssche, M.
Substituent Effects on Reactivity and Spectral Parameters
of Cephalosporins. J . Med. Chem. 1984, 27, 694-700.
3. Sugioka, T.; Asano, T.; Chikaraishi, Y.; Suzuki, E.; Sano, A.;
Kuriki, T.; Shirotsuka, M.; Saito, K. Stability and Degrada-
tion Pattern of Cefpirome (HR 810) in Aqueous Solution.
Chem. Pharm. Bull. 1990, 38, 1998-2002.
4
5
6
diate which rapidly formed an aldehyde as illustrated in
Scheme 2 using ceftazidime in place of cefpirome.13
Cephalosporin â-lactam hydrolysis produces cephalosporo-
2
1
ates which are generally too labile to isolate.
For
example, the hydrolysis of cefixime, which has a vinyl
group at C-3, formed an ethylidene cephalosporoate inter-
mediate which was similar to the exomethylene in Scheme
7
8
2
. The rate constant for conversion of that cephalosporoate
to the aldehyde was 500 times faster than the rate constant
for its formation.22
9
Thus, Scheme 2 represents the expected ceftazidime
hydrolysis products under current conditions on the basis
of the hydrolysis behavior for cephalosporins with suitable
leaving groups at the 3-methylene position. The observed
first-order behavior at each initial concentration required
that the total catalytic concentration did not change to a
kinetically distinguishable extent throughout the reaction.
Hydrolysis of the exomethylene intermediate gives rise
to the open-chain counterpart of the dihydrothiazine
moiety, which in turn degrades to products that have not
been previously characterized. This degradation is likely
to involve decarboxylation, thus resulting in the loss of the
1
1
1
1
Journal of Pharmaceutical Sciences / 57
Vol. 87, No. 1, January 1998

1
1
4. Connors, K. A. Simple Rate Equations. Chemical Kinetics;
VCH: New York, 1990; pp 18-21.
21. Kaiser, G. V.; Kukolja, S. Modifications of the â-Lactam
System. In Cephalosporins and Penicillins; Chemistry and
Biology, Flynn, E. H., Ed.; Academic Press: New York, 1972;
pp 125-128.
5. Yamaha, T.; Tsuji, A. Comparative Stability of Cephalospor-
ins in Aqueous Solution: Kinetics and Mechanisms of
Degradation. J . Pharm. Sci. 1976, 65, 1563-1573.
22. Namiki, Y.; Tanabe, T.; Kobayashi, T., Tanabe, J .; Okimura,
Y.; Koda, S.; Morimoto, Y. Degradation Kinetics and Mech-
anisms of a New Cephalosporin, Cefixime, in Aqueous
Solution. J . Pharm. Sci. 1987, 76, 208-214.
1
6. Faraci, W. S.; Pratt, R. F. Elimination of a Good Leaving
Group From the 3′-Position of a Cephalosporin Need Not Be
Concerted With â-Lactam Ring Opening: TEM-2 â-Lacta-
mase-Catalyzed Hydrolysis of Pyridine-2-azo-4′-(N′,N′-dimet
hylaniline) Cephalosporin (PADAC) and of Cephaloridine. J .
Am. Chem. Soc. 1984, 106, 1489-1490
23. Parker, V. Inverse Temperature Effects and Concentration
Dependent Apparent First-Order Rate Constants in Complex
Ion Radical Reactions. Acta Chem. Scand. B 1981, 35, 123-
129.
1
1
7. Itakura, K; Aoki, I; Kasahara, F; Nishikawa, M; Mizushima,
Y. Structures of Polymers Formed in Aqueous Solutions of
Cefsulodin Sodium. Chem. Pharm. Bull. 1981, 29, 1655-
1
661.
Acknowledgments
8. Boyd, D. B.; Lunn, W. H. W. Electronic Structures of
Cephalosporins and Penicillins. 9. Departure of a Leaving
Group in Cephalosporins. J . Med. Chem. 1979, 22, 778-783,
and refs 1-15 cited therein.
We thank Dr. Nagesh R. Palepu and SmithKline Beecham Inc.
for supplying ceftazidime. J osephine O. Fubara, recipient of the
Earl Higgins Fellowship, expresses appreciation for this support
and gratefully acknowledges receipt of the award for best podium
presentation at the seventh Annual Meeting of Graduate Students
In Pharmaceutics; J une 6-8, 1997, Gainesville, FL, based on this
work.
1
2
9. Wang, D.; Notari, R. E. Cefuroxime Hydrolysis Kinetics and
Stability Predictions in Aqueous Solutions. J . Pharm. Sci.
1
994, 83, 577-581.
0. Ceftazidime. In AHFS Drug Information 94; McEvoy, G. K.,
Ed.; American Society of Hospital Pharmacists Inc.: Be-
thesda, MD, 1994; pp 130-138.
J S970179K
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8 / Journal of Pharmaceutical Sciences
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