Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 72 No. 6 pp.

1253ñ1258, 2015 ISSN 0001-6837

Polish Pharmaceutical Society

THE RADIOLYTIC STUDIES OF CEFTRIAXONE IN THE SOLID STATE

PRZEMYS£AW ZALEWSKI1*, DARIA SZYMANOWSKA-POWA£OWSKA2, PIOTR GARBACKI1,
MAGDALENA PACZKOWSKA1, ALICJA TALACZY—SKA1 and JUDYTA CIELECKA-PIONTEK1

Department of Pharmaceutical Chemistry, Poznan University of Medical Sciences,

1

Grunwaldzka 6, 60-780 PoznaÒ, Poland

2
Department of Biotechnology and Food Microbiology, PoznaÒ University of Life Sciences,
Wojska Polskiego 48, 60-627 PoznaÒ, Poland

Abstract: The possibility of applying radiation sterilization to ceftriaxone disodium in the solid state was inves-
tigated. The lack of significant changes in the assay of ceftriaxone disodium irradiated with a dose of 25 kGy,
required to attain sterility, was confirmed. The antibacterial activity of ceftriaxone disodium irradiated with a
dose of 25 kGy, was unaltered for Gram-positive bacteria except of Staphylococcus aureus and changed for
Gram-negative strains with except of Klebsiella pneumoniae. Proteus vulgaris showed the greatest sensitivity to
CTD even after the application of radiation sterilization of 400 kGy.

Keywords: ceftriaxone disodium, radiation sterilization, stability

Ceftriaxone disodium (CTD) (Fig. 1) is a third- EXPERIMENTAL

generation semisynthetic cephalosporin with a long
half-life which has resulted in a recommended once Standards and reagents
daily administration schedule. It is administered Biotrakson is a sterile powder containing cef-
intravenously or intramuscularly and has a broad triaxone disodium, for preparation of injections
spectrum of activity against Gram-positive and (Bioton Sp. z o.o. Warszawa, Poland). All other
Gram-negative aerobic and some anaerobic bacteria. chemicals and solvents were obtained from Merck
CTD is effective in complicated and uncomplicated KGaA (Germany) and were of analytical grade.
urinary tract infections, lower respiratory tract infec- High quality pure water was prepared by using a
tions, skin, soft tissue, bone and joint infections, Millipore Exil SA 67120 purification system
septicemia and pediatric meningitis. In most of (Millipore, Molsheim, France).
above mentioned infections once-daily administra-
tion appears efficacious. Irradiation
CTD similarly to other cephalosporins have Twenty-five mg samples of CTD were placed
surprisingly few serious side effects, but the most of in 3 mL colorless glass vials that were closed with
them are caused by the generation of degradation plastic stoppers. The samples in the vials were
products. For example, fatal immune hemolytic ane- exposed to β irradiation in a linear electron acceler-
mia could be stimulated by a degradation product of ator LAE 13/9 (9.96 MeV electron beam and 6.2 µA
ceftriaxone (1) so it is important to estimate the sta- current intensity) until they absorbed doses of 25,
bility of cephems. The stability and mechanism of 50, 100, 200 and 400 kGy.
CTD degradation in aqueous solution (2) and in
solid state (3) were investigated in previous studies. Kinetic analysis
Other studies have confirmed that cephalosporins For the kinetic study, the Dionex Ultimate
are susceptible to degradation in aqueous solutions 3000 analytical system consisted of a quaternary
(4-11) and in the solid state (3, 12-18). Our investi- pump, an autosampler, a column oven and a diode
gations focused on determining the influence of array detector. As the stationary phase a Kinetex
radiation on CTD at doses of 25 (recommended for with 5 µm core-shell particles, C18, 100A, 100 ◊ 2.1
sterility) (19), 50, 100, 200 and 400 kGy. mm column was used. The mobile phase was com-

* Corresponding author: email: pzalewski@ump.edu.pl, phone: 0048-61-854-66-49

1253
1254 PRZEMYS£AW ZALEWSKI et al.

Figure 1. Chemical structure of ceftriaxone disodium

posed of acetonitrile ñ ammonium acetate (5 : 95, recovery level. The solutions were analyzed and the
v/v). The flow rate of the mobile phase was 1.0 percentages of recoveries were calculated.
mL/min and the injection volume was 10 µL. The
wavelength of the DAD detector was set at 254 nm. Precision
Separation was performed at 30OC. Precision of the assay was determined in rela-
The stability tests were performed according to tion to repeatability (intra-day) and intermediate
the International Conference on Harmonization precision (inter-day). In order to evaluate the
Guidelines (20). repeatability of the methods, six samples were
determined during the same day for three concen-
Method validation trations of CTD. Intermediate precision was studied
HPLC method was validated according to comparing the assays performed on two different
International Conference on Harmonization days.
Guidelines. The method was validated for parame-
ters such as specificity, linearity, precision, accura- Limits of detection (LOD) and quantification
cy and robustness. (LOQ)
The LOD and LOQ parameters were deter-
Selectivity mined from the regression equation of CTD: LOD =
The selectivity was examined for non-degrad- 3.3 Sy/a, LOQ = 10 Sy/a; where Sy is a standard error
ed and degraded samples in aqueous solutions in and a is the slope of the corresponding calibration
conditions of acid, base and neutral hydrolysis and curve.
in the solid state (thermal and radiolytic degrada-
tion). Robustness
The robustness of the procedure was evaluated
Linearity after changing the following parameters: the compo-
Linearity was evaluated in the concentration sition of the mobile phase; content of acetonitrile 5
range 42.4ñ508.8 mg/L (10-120% of the nominal ± 1 [%], the mobile phase flow rate 1.0 ± 0.2
concentration of CTD during degradation studies). [mL/min]; wavelength of detection 254 ± 5 [nm]
The samples of each solution were injected three and temperature 30 ± 2 [OC]. For each parameter
times and each series comprised 7 experimental change, its influence on the retention time, resolu-
points. tion, area and asymmetry of peak was evaluated. No
significant changes in resolution and shapes of peak,
Accuracy, as recovery test areas of peak and retention time were observed
The accuracy of the method was determined by when above parameters were modified. Modifi-
recovering CTD from the placebo. The recovery test cations of the composition of the mobile phase:
was performed at three levels 80, 100 and 120% of organic-to-inorganic component ratio resulted in the
the nominal concentration of CTD during degrada- essential changes of retention time and resolution in
tion studies. Three samples were prepared for each determination of CTD.
 The radiolytic studies of ceftriaxone in the solid state 1255

Microbiological analysis The concentrations of irradiated CTD were

Indicator microorganisms (Salmonella enteri- 0.02ñ256 µg/mL. Then, irradiated CTD of decreas-
tidis ATCC 13076, Salmonella typhimurium ATCC ing concentrations were added to each of test tubes.
14028, Listeria monocytogenes ATCC 7644, Next, test tubes were inoculated with the same
Staphylococcus aureus ATCC 25923, Escherichia amount of cells suspension. After 16-18 h of incu-
coli ATCC 25922, Pseudomonas aeruginosa ATCC bation at 37OC, the growth of strains was checked
27853, Proteus vulgaris ATTC 33420 and Klebsiella via turbidity increase observation. In test tubes con-
pneumoniae ATCC 31488) were cultured in soy- taining less than MIC of examined drugs the turbid-
casein broth with yeast extract for microorganisms ity increase was observed (the cells have grown).
with increased nutritional requirements. Clostridium The minimal concentration of drugs that inhibited
butyricum ATCC 860 and Clostridium pasterianum strain growth was defined as MIC.
ATCC 6013 were grown in Reinforced Clostridial
Medium (RCM, Oxoid, UK). Bacteria were cultured RESULTS AND DISCUSSION
under aerobic or anaerobic conditions (37OC, 24 h)
(depending on stains). Minimum inhibitory concen- The HPLC method for determination of CTD
tration (MIC) endpoints were determined by broth was found selective in the presence of degradation
microdilution according to CLSI guidelines (21). product as shown in Figuress. 2 and 3. In the chro-

Figure 2. The HPLC chromatogram of irradiated CTD (tR = 0.787 min) at doses of 25 kGy (recommended for sterility)

Figure 3. The HPLC chromatogram of irradiated CTD (tR = 0.787 min) at doses of 400 kGy
1256 PRZEMYS£AW ZALEWSKI et al.

Table 1. Intra-day, inter-day precision (n = 6) and recovery studies (n = 6).

Spiked concentration Measured concentration ± S.D.

(mg/L) (mg/L) RSD (%)
Intra-day precision
508.8 508.72 ± 0.02 0.0329
424.0 424.10 ± 0.06 0.0685
381.6 380.85 ± 0.04 0.0755
Inter-day precision
08.80 508.85 ± 0.07 0.0467
424.0 424.92 ± 0.08 0.0858
381.6 382.15 ± 0.06 0.1062
Recovery studies
Spiked concentration Measured concentration ± S.D. Recovery
(mg/L) (mg/L) (%)
508.8 (~ 120%) 509.11 ± 0.05 100.24
424.0 (~ 100%) 423.95 ± 0.03 99.62
381.6 (~ 80%) 381.59 ± 0.02 99.99

Table 2. Results of quantitative analysis of CTD before and after In contrast to the considerable instability of
irradiation. CTD under the influence of acid-basic hydrolysis
Dose [kGy] Content [%] (2), increased temperature and relative air humidity
0 100 (3), after exposure to a radiation dose of 25 kGy
25 97 CTD only 3% from initial concentration of CTD
degraded (Table 2). A comparison of HPLC study
50 94
results for non-irradiated and irradiated CTD sam-
100 93 ples demonstrated that the concentration of CTD
200 91 changed after irradiation in higher doses (Table 2).
400 80 The effectiveness of radiation sterilization and
possible changes in microbiological activity were
also evaluated. The results of microbiological studies
proved that irradiation at 25 kGy ensured the sterili-
matograms of CPS developed over a period of 0 to ty of CTD samples. All tested species of reference
5 min the following compounds were eluted: CTD strains showed sensitivity to CTD, both when non-
with a retention time of 0.787 min and degradation sterilized and following different irradiation doses
product with retention time of 0.33 min. Peaks were (Table 3). By comparing the activity of non-irradiat-
symmetrical, clearly separated from each other ed and irradiated CTD samples it was found that no
(Figs. 2 and 3). The calibration plots were linear in significant differences between MIC values for the
the following concentration range 42.4ñ508.8 mg/L reference Gram-positive strains (Listeria monocyto-
(n = 11, r = 0.9999). The calibration curve was genes, Clostridium butyricum and Clostridium pas-
described by the equation y = ax; y = (0.404 ± teurianum) and Gram-negative Klebsiella pneumo-
0.002) x. The b value, calculated from equation y = nia (Table 3) occur. On the other hand, CTD is active
ax + b, was not significant. Statistical analysis against Listeria monocytogenes and Klebsiella pneu-
using Mandelís fitting test confirmed linearity of monia only at high concentrations (Table 3). In the
the calibration curves. The intra-day and inter-day case of the reference strains (Salmonella enteritidis,
precision values of measured concentration of Salmonella typhimurium, Staphylococcus aureus,
CTD, as calculated from linearity plots are given in Escherichia coli, Pseudomonas aeruginosa and
Table 1. The RSD values were from 0.0329 to Proteus vulgaris), the bactericidal properties of irra-
0.1062% demonstrating that the method was pre- diated CTD samples were substantially reduced
cise. The LOD and LOQ were 2.5 and 7.5 mg/L, (Table 3). For Staphylococcus aureus even no inhibi-
respectively. tion of growth was observed. Among all the analyzed
 The radiolytic studies of ceftriaxone in the solid state 1257

Table 3. MIC values (Ïg/mL) of irradiated CTD samples.

Reference strain CTD CTD CTD

0 kGy 25 kGy 400 kGy
Gram-negative bacteria
Salmonella enteritidis ATCC 13076 0.24 32 32
Salmonella typhimurium ATCC 14028 0.24 32 32
Escherichia coli ATCC 25922 1 8 32
Klebsiella pneumoniae ATCC 31488 32 32 > 256
Pseudomonas aeruginosa ATCC 27853 64 128 256
Proteus vulgaris ATCC 33420 0.12 1 4
Gram-positive bacteria
Clostridium butyricum ATCC 860 4 4 16
Clostridium pasterianum ATCC 6013 4 4 16
Staphylococcus aureus ATCC 25923 8 > 256 > 256
Listeria monocytogenes ATCC 7644 64 64 256

strains of the reference bacteria Proteus vulgaris CTD limits the application of radiation sterilization
showed the highest sensitivity to CTD, even in the as an alternative method to obtain sterile dosage
variant, in which the irradiation dose applied on the forms of CTD. Changes in the concentration of CTD
investigated compound was 400 kGy. The difference irradiated at 25 kGy cause a decrease in its microbi-
connected with the sensitivity of Gram-negative ological activity against Gram-negative strains.
(Pseudomonas aeruginosa) and Gram-positive bac- Although that dose of radiation does not lead to any
teria (Clostridium sp.) could have resulted from the significant differences in the assay of CTD (3%), its
differences connected with cell wall structure, partic- bactericidal activity against Salmonella enteritidis,
ularly since the bactericidal activity of CTD results Salmonella typhimurium, Staphylococcus aureus,
from the inhibition of bacterial cell wall synthesis. Escherichia coli, Pseudomonas aeruginosa and
Moreover, as a result of the effect of radiation steril- Proteus vulgaris was decreased.
ization, the spatial conformation of the compound
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Received: 26. 09. 2014