Stability of aztreonam in AZACTAM

Article abstract of DOI:10.1016/j.farmac.2005.04.009

The influence of temperature and relative humidity on the stability of aztreonam in AZACTAM was investigated. Changes of the concentration of aztreonam were followed using the HPLC method with UV detection. The first-order rate constants of the reversible reaction of isomerization Z-aztreonam ? E-aztreonam and the parallel reaction Z-aztreonam → products were determined at RH = 76.4% and T = 313, 323, 333, 343 and 353 K, and at T = 343 K and RH = 50.9%, 60.5%, 66.5% and 76.4%. The thermodynamic parameters-energy, enthalpy and entropy of these reactions were calculated.

Full text of DOI:10.1016/j.farmac.2005.04.009

Il Farmaco 60 (2005) 599–603
http://france.elsevier.com/direct/FARMAC/
Stability of aztreonam in AZACTAM
Marianna ZajTc a,*, Anna Jelin´ska a, Judyta Cielecka-Piontek a, Irena Oszczapowicz b
a Department of Pharmaceutical Chemistry, University of Medical Sciences in Poznan´, Poznan´, Poland
b Department of Modified Antibiotics, Institute of Biotechnology and Antibiotics, Warszawa, Poland
Received 10 November 2004; revised and accepted 21 April 2005
Available online 01 June 2005
Abstract
The influence of temperature and relative humidity on the stability of aztreonam in AZACTAM was investigated. Changes of the concen-
tration of aztreonam were followed using the HPLC method with UV detection. The first-order rate constants of the reversible reaction of
isomerization Z-aztreonam d E-aztreonam and the parallel reaction Z-aztreonam → products were determined at RH = 76.4% and T = 313,
323, 333, 343 and 353 K, and at T = 343 K and RH = 50.9%, 60.5%, 66.5% and 76.4%. The thermodynamic parameters—energy, enthalpy
and entropy of these reactions were calculated.
© 2005 Elsevier SAS. All rights reserved.
Keywords: Aztreonam; Stability in solid phase; Kinetic and Thermodynamic parameters
1. Introduction
Ni(II) and Mn(II) [4], photodegradation of aztreonam under
UV light [5], the stability of aztreonam in 5% dextrose and
0.9% sodium chloride injection [6] were investigated.
The purpose of this study was to evaluate the stability of
aztreonam AZACTAM in an atmosphere of increased rela-
tive humidity.
Aztreonam is the first antibiotic from the monobactam fam-
ily to have been therapeutically approved. It was introduced
in 1987 in the form of injections in the USA and is now used
in most European countries in the treatment of infections
against Gram-negative bacteria.
Aztreonam is derived of
3-aminomonobactamic acid
(3-AMA). The sulfonic acid
substituent in the 1-position
of the ring activates the beta-
lactam moiety, while the ami-
nothiazolyl oxime side chain
in the 3-position and the
methyl group in the 4-posi-
tion ensures beta-lactamase
stability [1,2].
2. Experimental
2.1. Material and reagents
Azactam—a sterile, nonpyrogenic, sodium-free, white
powder containing approximately 780 mg arginine per g of
aztreonam for intramuscular or intravenous use (E.R. Squibb
and Sons Ltd.). Diprophylline suitability with FP VI.
Buffer salts used and other chemicals were commercial
products of analytical grade.
In previous studies, the general and specific acid–base
catalysis over a pH range from 3.50 to 10.50 at 35 °C [3], the
decomposition of aztreonam in aqueous solution of Tris at a
constant temperature (35 °C) and ionic strength (0.5 mol l–1)
in the presence of metal ions (Zn(II), Cd(II), Co(II), Cu(II),
2.2. Analytical procedure
The method used in the experiments is a modification of
the procedure presented in USP 24.
The liquid chromatograph was equipped with an
L-6000 pump, an LC-2UV detector (Shimadzu) and a Rheo-
dyne 7120 (California, USA) with a 20 µl fixed-loop injector.
* Corresponding author. Tel.: +48 618 546 651; fax: +48 618 546 652.
E-mail address: mzajac@amp.edu.pl (M. ZajTc).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.04.009

600
M. ZajTc et al. / Il Farmaco 60 (2005) 599–603
A Merck (Darmstadt, Germany) analytical column (LiChro-
spher RP-18, 5 µm particle size, 25 cm × 4 mm, ID) was used
as the stationary phase. The mobile phase consisted of metha-
nol and phosphate buffer (1:4) (6.8 g KH2PO4 dissolved in
water to make 1000 ml, and adjusted with 1 mol l–1 H3PO4 to
a pH of 3.0 0.1). The flow rate was 1.0 ml min–1. UV detec-
tion was set at 270 nm. All chromatographic operations were
conducted under ambient conditions.
2.3. Conditions of the kinetic studies
For the experiments 10 mg samples of Azactam were
weighed into 5 ml vials. Samples tested for the influence of
temperature in a humid environment were placed in desicca-
tors containing saturated solutions of sodium chloride (RH ~
76.4%) inserted in heat chambers set to the desired tempera-
tures 313, 323, 333, 343 and 353 K. In order to examine the
influence of humidity, samples were placed in desiccators con-
taining saturated solutions of inorganic salts which ensured
the desired relative humidity of the ambient air [8] (sodium
bromide—RH = 50.9%, potassium iodide—RH = 60.5%,
sodium nitrate—RH = 66.5% and sodium chloride—RH =
76.4%) inserted in heat chambers set to 343 K.
At definite time intervals, determined by the rate of deg-
radation, the vials were removed, cooled to room tempera-
ture and the contents dissolved in a mixture of methanol and
water (1:4 v/v). The so obtained solutions were quantita-
tively transferred into measuring flaks and completed to a
total volume of 10.0 ml the above-mentioned solvents. To
1.0 ml of the so obtained solution 1.0 ml of a solution of
internal standard (diprophylline (0.4 mg ml–1) in a mixture
(1:4) of methanol and water) was added. Twenty microliters
samples of the solutions were injected onto the column. The
method was validated (selectivity, linearity, precision and lim-
its of detection and quantitation) to assess its suitability for
analyzing the stability of aztreonam in AZACTAM.
The values h/hIS (h and hIS—heights of aztreonam and
internal standard peaks, respectively) were used to interpret
the changes of concentration of Z-and E-aztreonam during
the reaction.
Fig. 1. HPLC chromatogram of the Azactam after 7 h heating at 333 K and
RH = 76.4%; 1 – Z-aztreonam, 2 – E-aztreonam, IS – internal standard (dipro-
phylline), P – product.
3.1.2. Linearity
The linearity between h/hIS (h and hIS—heights of aztre-
onam and IS peaks) and the concentration of aztreonam in a
mixture of methanol and water (1:4) ranging from 0.0702
to 0.9831 mg ml–1 was evaluated. The linear dependence
h/hIS = f(c) was described by the equation y = a · c =
(2.126 0.060) · c (S.D. = 0.027, Sy = 0.0299, r = 0.9988 for
n = 14 and ␣ = 0.05).
3.1.3. Limits of detection and quantitation
The limits of detection (DL = 0.0464 mg ml–1) and quan-
titation (QL = 0.141 mg ml–1) were calculated from the for-
mulas DL = 3.3 Sy/a and QL = 10 Sy/a.
Microsoft® Excel 2000 was used for the calculation of
regression parameters
a
Da and b Db in the equation y = ax + b, standard
3.1.4. Precision
deviation Sa, Sb, Sy and the coefficient of linear correlation r.
The values a Da and b Db were obtained for f = n – 2
degrees of freedom with ␣ = 0.05.
The precision of the determination of aztreonam was esti-
mated for three concentration of the standard solution ofAzac-
tam: 0.5, 1.0 and 1.5 mg ml–1 in a mixture (1:4) of methanol
and water. The following results were obtained for the above
concentration for n = 8 and ␣ = 0.05: x1 = 0.5944,
S.D. = 0.0039, R.S.D. = 0.66%, Sy = 0.014; x2 = 1.2088,
S.D. = 0.0015, R.S.D. = 0.13%, Sy = 0.013; x3 = 1.7112,
S.D. = 0.0012, R.S.D. = 0.07%, Sy = 0.0004.
3. Investigations and results
3.1. Validation of the HPLC method
3.1.1. Selectivity
3.2. The kinetics of the degradation of aztreonam
The applied method was selective for Z-aztreonam
(tR = 4.20 min), E-aztreonam (tR = 2.73 min), internal stan-
dard (diprophylline, tR = 4.68 min) and the degradation prod-
uct (tR = 5.80 min) (Fig. 1).
3.2.1. Rate constants
The changes of the concentration of substrate and prod-
ucts (Fig. 2) show that the decomposition of aztreonam in

M. ZajTc et al. / Il Farmaco 60 (2005) 599–603
601
Fig. 2
.
Semilogarythmic plots of hi/hIS = f (t) for E-aztreonam, Z-aztreonam and product (P) at 333 K and RH = 76.4%.
AZACTAM in an air of increased relative humidity is thought
to be a consequence of the reaction illustrated in Scheme 1,
where k1 is the isomerization rate constant from Z-isomer to
E-isomer, and k–1 is the reverse, and k2 i k3 are the degrada-
tion rate constants of Z- and E-isomers.
values c′i for time ti were calculated by the extrapolation of
the linear dependence ln c = f (t) in the time range te → t∞.
The value c′0 for t = 0 denotes the concentration of Z-isomer
in a state equilibrium.
The partial rate constants k1 and k–1 (Table 1) of the revers-
ible reaction were obtained from the following relationships
k–1 = ks/(1 + K) and k1 = ks – k–1.
Similar mechanism of the degradation of aztreonam was
observed in aqueous solution at pH 2.5 [7].
According to Scheme 1, the following kinetic equations
can be obtained for Z- and E-isomers:
The equilibrium constant (K) was calculated from the equa-
tion [8]:
K = k1/k–1 = [E-aztreonam]e/[Z-aztreonam]e = (c0 – ce)/ce
The ratio E-aztreonam/Z-aztreonam was approximately
0.6 at 333 K and RH = 76.4%.
d[Z] ⁄ d t = −k1[Z] + k−1[E] − k2[Z]
d[E]/ d t = k1[Z] − k−1[E] − k3[Z]
The similar dependence was observed in the case of
E-isomer. The slope of the plot ln c = f(t) in the time range te
→ t∞ equals –k3 and the slope of the remainder plot ln (c′i –
ci) = f (t) in the time range t0 → te equals –ks. The rate con-
stant k3 was smaller than k2; at 333 K and RH = 76.4%,
k2 = 5.73 × 10–6 s–1 and k3 = 1.35 × 10–6 s–1.
The first-order rate constants k2 (Table 1) were computed
for the values of Z-aztreonam concentration in the time range
te → t∞, in which the linear dependence ln ci = f (t) was
observed (Figs. 3 and 4).
The rate constants ks = k1 + k–1 (Table 1) of the reversible
reaction Z-aztreonam d E-aztreonam were determined from
the remainder plots ln (ci – c′i) = f(t) (Figs. 5 and 6) in the
time range t0 → te. The slopes of these plots a = –ks. The
3.2.2. The effect of temperature
The influence of temperature on the degradation of
Z-aztreonam inAZACTAM is described by theArrhenius rela-
tionship ln ki = ln A + a (1/T). The value ln A (A = frequency
coefficient) and a (the slope of the plot ln ki = f (1/T)) were
used to calculate energy (Ea), enthalpy (DH≠) and entropy
(DS≠) of the reaction (Table 2) from appropriate equations
[8].
Scheme 1
.
Table 1
The first-order rate constants of the degradation of aztreonam in AZACTAM
Parameters
T (K)
313
(k2 Dk) (s–1
)
(ks Dk) (s–1
)
k1 (s–1
)
K–1 (s–1
)
RH = 76.4%
(8.48 0.03) × 10–7
(2.23 0.38) × 10–6
(5.73 0.42) × 10–6
(9.32 2.39) × 10–6
(3.36 0.70) × 10–5
T = 343 K
(9.58 0.01) × 10–5
(2.39 0.29) × 10–4
(6.34 1.06) × 10–4
(1.62 0.26) × 10–3
(2.69 1.23) × 10–3
3.24 × 10–5
8.45 × 10–5
2.11 × 10–4
5.57 × 10–4
8.91 × 10–4
6.34 × 10–5
1.55 × 10–4
4.24 × 10–4
1.07 × 10–3
1.81 × 10–3
323
333
343
353
RH%
50.9
(1.12 0.58) × 10–6
(4.10 0.62) × 10–6
(5.00 0.55) × 10–6
(9.32 0.24) × 10–6
(2.20 0.85) × 10–4
(3.64 0.15) × 10–4
(5.25 0.12) × 10–4
(16.2 0.26) × 10–4
0.554 × 10–4
1.54 × 10–4
2.17 × 10–4
5.57 × 10–4
1.65 × 10–4
2.10 × 10–4
3.12 × 10–4
10.7 × 10–4
60.5
66.5
76.4

602
M. ZajTc et al. / Il Farmaco 60 (2005) 599–603
Table 2
The thermodynamic parameters of the degradation of aztreonam in AZACTAM
Parameters
k2
ks
k1
k–1
ln ki = f (1/T)
a = –9770 1979
Sa = 622
a = –9540 1659
Sa = 521
a = –9460 1832
Sa = 576
a = –9598 1596
Sa = 502
b = 17.20 5.50
Sb = 1.87
b = 21.23 4.36
Sb = 1.57
b = 19.92 5.20
Sb = 1.73
b = 21.00 4.43
Sb = 1.51
r = –0.9939
Sy = 0.1771
77.9 11.0
75.5 13,55
–112 212
r = –0.9945
Sy = 0.1485
79.4 11.9
76.9 14.4
–68 209
r = –0.9944
Sy = 0.1639
78.7 13.3
76.2 15.8
–79 205
r = –0.9959
Sy = 0.1428
79.8 11.6
77.3 14.1
–70 211
Ea (kJ mol–1
DH≠ (kJ mol–1
DS≠ (J/(mol . K))
)
)
n, number of experiments; Dk = Sa t␣f; Ea, activation energy; DH≠, enthalpy; DS≠, entropy; Ea = –a R (J mol–1); DH≠ = Ea – RT (J mol–1); DS≠ = R(ln A – ln
(kBT)/h), where: kB stands for the Boltzmann constant (1.3807 × 10–23 J K–1); h, Plancks constant (6.6256 × 10–24 J s); R, universal gas constant
(8.314 J K–1 mol–1); T, temperature in K (t + 273 K); a, vectorial coefficient of the Arrhenius relationship and A, stands for the frequency coefficient.
Fig. 5
. Semilogarythmic plots of ci – c′i = f (t) of the degradation of the aztreo-
nam in Azactam at different temperatures (RH = 76.4%).
Fig. 3
. Semilogarythmic plots of ci = f (t) of the degradation of the aztreo-
nam in Azactam at different temperatures (RH = 76.4%).
Fig. 6. Semilogarythmic plots of ci – c′i = f (t) of the degradation of the aztreo-
nam in Azactam at various humidities at 358 K.
4. Conclusions
The degradation of Aztreonam in AZACTAM in the air of
increased relative humidity (50.9–76.4%) and temperature
(313–353 K) is the effect of the parallel reaction (Scheme 1)
as in the case of aqueous solutions at pH 2.5 [7]. However,
the reversible reaction of isomerization Z-aztreonam d
E-aztreonam is more important. The rate of this reaction is
about 100 times greater than that of the parallel reaction
Z-aztreonam → products, while the rate of the latter reaction
is about four times greater than that of the subsequent reac-
tion
Fig. 4. Semilogarythmic plots of ci = f (t) of the degradation of the aztreo-
nam in Azactam at various humidities at 358 K.
3.2.3. The effect of humidity
The effect of humidity (RH > 50%) on the stability of aztre-
onam inAZACTAM is described by the following equations:
lnk1 = (7.31 5.15)10−2 · RH% − 14.3 1.0; r = 0.9960
lnk−1 = (8.87 1.56)10−2 · RH% − 12.7 3.3; r = 0.9414
lnk2 = (8.03 4.30)10−2 · RH% − 17.6 2.8; r = 0.9647
E-aztreonam → products.
The thermodynamic parameters of each reaction (Table 2)
as well as the effect of the relative air humidity in the range

M. ZajTc et al. / Il Farmaco 60 (2005) 599–603
603
[2] M. ZajTc, E. Pawełczyk, Pharmaceutical Chemistry, University of
Medical Sciences in Poznan´, 2000 (in Polish).
50.9–76.4% on the rate of each reaction, do not show statis-
tically significant differences.
[3] R. Méndez, T. Alemany, J. Martin-Villacorta, Stability in aqueous
solution of two monocyclic b-lactam antibiotics: aztreonam and
nocardicin A, Chem. Pharm. Bull. (Tokyo) 40 (1992) 3222–3227.
[4] R. Méndez, T. Alemany, J. Martin-Villacorta, Catalysis of hydrolysis
and aminolysis of non-classical b-lactam antibiotics by metal ions and
metal chelates, Chem. Pharm. Bull. (Tokyo) 40 (1992) 3228.
[5] H. Fabre, H. Ibork, D.A. Lerner, Photodegradation kinetics under UV
light of aztreonam solutions, J. Pharm. Biomed. Anal. 10 (1992) 645.
[6] L.A. Trissel, Q.A. Xu, J.F. Martinez, Compatibility and stability of
aztreonam and vancomycin hydrochloride, Am. J. Health Syst.
Pharm. 52 (1995) 2560.
Acknowledgements
This study was supported by a research grant from the Uni-
versity of Medical Sciences in Poznan´, Poland (no. 501-3-
0000225).
References
[7] F. Bruchhausen, S. Ebel, A.W. Frahm, E. Hackenthal, Hagers Hand-
buch der Pharmazeutischen Praxis, Springer-Verlag, Berlin, 1993.
[8] E. Pawełczyk, T. Hermann, The Fundamentals of the Stability of
Drugs, PZWL, Warszawa, 1982 (in Polish).
[1] Bristol-Myers Squibb Company, Princeton, NJ 08543, USA; Informa-
tion of AZACTAM®.