H-J Mller1, K Howe2, C Frank3, I Haker4 1 Director Pharmaceutical Product Support, Pharma Division, Innovation Centre, Fresenius AG,

Germany; 2Quality Assurance Manager, Fresenius Ltd, Great Britain; 3Scientific Collaborator, Pharmaceutical Product Support, Pharma Division, Innovation Centre, Fresenius AG, Germany; 4Laboratory Manager, Analytical Research, Pharma Division, Innovation Centre, Fresenius AG, Germany

STABILITY OF CEFAZOLIN, CEFOTIAM, CEFUROXIME, CEFOTAXIME, CEFTRIAXONE AND CEFTAZIDIME IN NORMAL SALINE SOLUTIONS, STORED IN A NEW I.V. CONTAINER MADE OF BIOFINE
The stability of six cephalosporins was studied when diluted and stored in a modified, new-generation polypropylene flexible container. Commonly used dosages of cefazolin sodium, cefotiam hydrochloride, cefuroxime sodium, cefotaxime sodium, ceftriaxone disodium, and ceftazidime dissolved in 0.9% sodium chloride were stored both at room temperature and under refrigeration. The solutions were also stored in glass bottles as controls. Drug concentrations were measured by stability-indicating HPLC. The physical parameters of: pH, UV absorbance, appearance, and subvisible particles were determined. Stability was defined as retention of at least 90% of the original drug concentration unless there were any known toxicity problems associated with the breakdown products. The results of the studies for both the new flexible container and the glass bottle controls correlate with the literature. No influence of the novel packaging material Biofine could be detected on the stability of the investigated cephalosporins when compared with the results of the glass bottles. KEY WORDS: Cephalosporins, stability, compatibility, PVC-free bags

INTRODUCTION Cephalosporins are often used to treat infections with gram-negative and gram-positive micro-organisms as well as in perioperative prophylaxis [1]. For this reason, the stability of cefazolin (a first generation cephalosporin), cefotiam and cefuroxime (second generation cephalosporins), and cefotaxime, ceftriaxone and ceftazidime (third generation cephalosporins) were tested in the new container. Fresenius AG has developed Biofine, a new material that combines the advantages of each available traditional packaging system. The product is flexible, transparent, lightweight and uses polyolefine materials only. The primary container has been developed to meet all of the physical and mechanical requirements of the third edition of the European Pharmacopoeia Plastic containers for aqueous solutions in parenteral infusion [2] and additional specifications of the German standard DIN 58363 [3]. The major benefits of the material include its enhanced drug compatibility, clarity, flexibility, ease of recycling, and a major reduction of extractable material when compared with traditional flexible containers. Figure 1 shows the chemical structure of the cephalosporins studied, with the general routes of breakdown shown in Figure 2. Figure 1. Cephalosporin structures. Figure 2. Generalized method of hydrolytic breakdown and rearrangement of cephalosporins. Figure 3. Decrease in active concentration of the six cephalosporins studied with time at both room temperature and refrigerated (28C) in the new container and glass bottles. a=cefazolin; b=cefuroxime; c=cefotiam; d=ceftriaxone; e=ceftazidime; f=cefotaxime; new container material; glass bottle Figure 4. Decrease in concentration of six cephalosporins with time to enable determination of the rank order of stability, for two temperatures used, for the new container. a=cefazolin; b=cefuroxime; c=cefotiam; d=ceftriaxone; e=ceftazidime; f=cefotaxime

figure 1

figure 2

figure 3

figure 4

The primary mechanism of breakdown is that of hydrolysis. Cephalosporins have two primary sites for hydrolysis; the substituted C3 position and the beta lactam ring [4]. Hydrolysis at these two sites is known to occur either alone or in parallel depending on the pH, temperature, ionic strength, etc., of the solvent present. Following cleavage at C3 it is usual to observe an internal rearrangement to form an internal ester i.e. a lactone. The shelf lives obtained for the named cephalosporins, in the same infusion fluid, will primarily revolve around the susceptibility of the individual cephalosporins to hydrolysis. This in turn will depend on the degree of shielding of the sites, the nature of the bond to be cleaved, the degree of polarization of the site and the inherent stability of the breakdown products. Under normal physiological conditions, hydrolysis of the amide bond present does not occur. As hydrolysis is the major route of breakdown, there should not be any major influence from the new container material. The new material will only have an effect if it either catalyses the hydrolysis process or if it physically interacts with the drug by mechanisms such as adsorption, absorption or permeation phenomena not usually associated with polypropylene. Direct comparison with glass containers will identify any such interactions. The purpose of this study was to determine the stability of six cephalosporins when prepared in the new container. This data was compared with control results generated in glass bottles. Investigation time plans were prepared according to available literature. METHODS Sample preparation Under aseptic conditions, approximately 10 mL sodium chloride 0.9% were removed from the new container (Fresenius AG, Biofine, Lot HAFE 20, Germany)/glass bottle (Fresenius AG, Lot GL 1001, Germany). The drugs (concentrations given in Table 1) were reconstituted in this volume and the resultant clear solutions reinjected. Admixtures were stored at room temperature as well as in a refrigerator (28C) protected from light. After fixed time intervals, the UV absorbance, pH, subvisible

particle counts, visual appearance and the content of the active substance were determined.

Table 1. Physical investigations.

Storage conditions Investigation time Cefazolin (10 mg/mL) Cefotiam (20 mg/mL) Cefuroxime (7.5 mg/mL) Cefotaxime (20 mg/mL) Ceftriaxone (20 mg/mL) Ceftazidime (20 mg/mL) room temperature 4C room temperature 4C room temperature 4C room temperature 4C room temperature 4C room temperature 4C 7 days 28 days 1 day 7 days 7 days 28 days 2 days 20 days 14 days 28 days 2 days 14 days 5.126.24 5.085.91 6.286.41 6.366.54 6.677.83 6.627.58 5.204.68 5.094.71 6.557.27 6.556.99 6.546.96 6.507.03 0.0320.024 0.0290.020 0.5203.234 0.8212.324 0.2133.214 0.2030.901 0.4041.393 0.5541.788 0.4543.639 0.4542.719 0.2990.431 0.3110.702 no increase during investigation time no increase during investigation time no increase during investigation time no increase during investigation time no increase during investigation time no increase during investigation time clear and colourless clear / light yellow yellow clear / light yellow yellow clear and light yellow clear / light yellow orange-brownish clear / light yellow yellow PH value Absorbance Particle Appearance

Table 2. Concentration at room temperature, Containermaterial Cefazolin (10 mg/mL) Cefotiam (20 mg/mL) Cefuroxime (7.5 mg/mL) Cefotaxime (20 mg/mL) Ceftriaxone (20 mg/mL) Ceftazidime (20 mg/mL) Biofine

Investigation Time
Initial 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 98.9% 98.8% 98.2% 98.7% 6 hours 99.7% 99.6% 95.8% 94.8% 89.82% 89.12% 12 hours 24 hours 99.4% 99.6% 82.3% 82.0% 93.4% 94.2% 92.6% 92.6% 101.6% 99.0% 94.9% 94.2% 86.1% 85.9% 85.0% 84.2% 100.2% 98.4% 90.3% 90.0% 98.5% 95.3% 2 days 98.5% 98.6% 5 days

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle

Table 3. Concentration at 28C. ContainerMaterial Cefazolin mg/mL) Cefotiam mg/mL) Cefuroxime mg/mL) Cefotaxime mg/mL) Ceftriaxone mg/mL) Ceftazidime mg/mL) (20 (20 (20 (7.5 (20 (10 Biofine Investigation Time Initial 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 1 day 99.8% 100.0% 90.8% 91.0% 96.3% 98.0% 96.2% 96.4% 99.7% 100.3% 100.4% 100.1% 89.4% 90.9% 88.2% 87.8% 91.1% 93.3% 87.5% 88.3% 99.4% 100.7% 95.2% 95.4% 92.7% 92.2% 97.5% 99.7% 89.2% 89.6% 96.0% 97.4% 95.0% 97.4% 84.5% 86.5% 4 days 7 days 100.1% 100.3% 10 days 99.5% 99.4% 14 days 21 days 28 days 96.7% 96.6%

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle Biofine

Glass Bottle

Table 4. Rank order of observed stability from Figure 4: 1= most stable.

Rank order of stability Cephalosporin Refrigerated (28C) Room temperature

Cefazolin Ceftriaxone Ceftazidime Cefuroxime Cefotaxime Cefotiam

1 2 3 4 5 6

1 2 3 4 5 6

Physical investigations The determinations of the UV absorption were performed using an UV/VIS double-beam spectrophotometer (Jouan GmbH, Hitachi U2000, Germany), in accordance to Ph. Eur. III, 2.2.25. The total spectral range (200800 nm) was measured. If no irregular values were observed, only the measured value at 400 nm was reported. The measurements were carried out against water as the reference solution. 40 mm quartz cuvettes were used. The determination of pH was carried out using a pH meter (WTW, pH 537, Type E 56, Germany) (Ph. Eur. 2.2.3.). The examination of subvisible particulate matter was in accordance with USP 23/NF 18<788> using a light blockage particle-counter (HIAC-ROYCO, 3000A, 9064, Germany). The number of particles 10 m/mL and 25 m/mL was determined. The solutions met the requirements of the test if the average numbers of particles present in the units tested did not exceed the limit values of 60 counts/mL 10 m and 6 counts/mL 25 m. The visual examinations were in addition to those for the subvisible particulate matter. The following visual changes were sought: opalescence/opacity, precipitation, gas bubble generation and any discoloration. Assays The contents of cefazolin (Fresenius AG, cephazolin Fresenius, Lot GI1700, Germany) cefotiam (Takeda Pharma GmbH, Spizef, Lot 897AB2, Germany), cefuroxime (Hoechst AG, Zinazef, Lot 6N034, Germany), cefotaxime (Hoechst AG, Claforan, Lots N132 and N079, Germany), ceftriaxone (Roche AG, Rocephin, Lots 59662 and 58262, Germany), and ceftazidime (Glaxo Wellcome, Fortum, Lot 7B948, Germany) were determined by high performance liquid chromatography (HPLC). The optimal conditions for the HPLC system were determined from preliminary degradation studies. Each assay was drug specific and distinguished the target antibiotic from its own breakdown products. The measurements of cefazolin, cefotiam, and ceftriaxone were carried out using equipment from Thermo-Separation-Products (Thermo Quest, Thermo Separation Products, P4000-Pump, AS2000 Autosampler, UV2000-Detector, Germany) including fast scan UV detector, with PC 1000 as control

and analysis software. The measurements of cefuroxime, cefotaxime, and ceftazidime were carried out using equipment from Hewlett Packard (Hewlett Packard, HP1100 with ChemStation, Germany) with DAD and Chem Station as control and analysis software. Cefazolin The content of cefazolin was determined by an internal HPLC method. A reversed phase column (Merck AG, LiChrospher 60 RP select B, Lot 1.50829, Germany) was used with an eluent of disodium hydrogen phosphate buffer and acetonitrile (83:17), flow rate of 1.0 mL/min. The samples were detected at 272 nm. The cefazolin content was determined after 0, 6, 24, 48 hours and after 7 days at room temperature, as well as after 0, 1, 7, 10, and 28 days under refrigeration (28C). Before each test, the refrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 50150 mg cefazolin/L (Tiefenbacher, standard cefazolin sodium, Lot EL 1700, Germany). The selectivity was detected by injecting a sample of 0.9% normal saline and checking for deviations in the baseline. No deviations were observed. The reproducibility of cefazolin was measured using six injections. This determination gave a precision of SD (%) < 1%. The day-to-day standard deviation over more than 10 days was SD (%) < 1%. Cefotiam The content of cefotiam was determined by an HPLC method in accordance with the USP 23 Monograph. A reversed phase column (Merck AG, LiChrospher 100 RP 18, Lot 1.50828, Germany) was used with an eluent of ammonium sulphate solution and acetonitrile (85:15), pH-value 6.5 (adjusted with ammonia), flow rate of 1.5 mL/min. The samples were detected at 254 nm. The cefotiam content was determined after 0, 6, 12, and 24 hours at room temperature and after 0, 1, and 4 days under refrigeration (28C). Before each test the refrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 2575 mg cefotiam/L (Grnenthal GmbH, standard cefotiam HCl, Lot 5G7262, Germany). The selectivity was detected by injecting a sample of 0.9% normal saline and checking for deviations in the baseline. No deviations were observed. The reproducibility of cefotiam was measured using six injections. This determination gave a precision of SD (%) < 1%. The day-to-day standard deviation over 7 days was SD (%) < 1.5%. Cefuroxime The content of cefuroxime was determined by an internal HPLC method. A reversed phase column (Merck AG, LiChrospher 60 RP select B, Lot 1.50829, Germany) was used with an eluent of dipotassium hydrogen phosphate buffer and acetonitrile (83:17), flow rate of 1.0 mL/min. The samples were detected at 260 nm. The cefuroxime content was determined after 0, 1, and 2 days at room temperature, as well as after 0, 1, 7, and 14 days under refrigeration (28C). Before each test the refrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 2575 mg cefuroxime/L (Tiefenbacher, Standard cefuroxime sodium, Lot 4409100665, Germany). The selectivity was detected by injecting a sample of 0.9% normal saline and checking for deviations in the baseline. No deviations were observed. The reproducibility of cefuroxime was measured using six injections. This determination gave a precision of SD (%) < 1%. The day-to-day standard deviation over 7 days was SD (%) < 1%. Cefotaxime The content of cefotaxime was determined by an HPLC method according to the USP 23 Monograph Sterile cefotaxime sodium. A reversed phase column (Merck AG, LiChrospher 100 RP 18, Lot 1.50828, Germany) was used with an eluent of buffer (containing KH2PO4, Na2HPO4, and H2O) and methanol (83:17), flow rate of 1.0 mL/min. The samples were detected at 254 nm. The cefotaxime content was determined after 0, 6, 24, and 48 hours at room temperature, as well as after 0, 1, 4, and 7

days under refrigeration (28C). Before each test the refrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 50150 mg cefotaxime/L (Promochem, CRS standard cefotaxime sodium, Lot 1, Germany). The selectivity was detected by injecting a sample of 0.9% normal saline and checking for deviations in the baseline. No deviations were observed. The reproducibility of cefotaxime was measured using six injections. This determination gave a precision of SD (%) < 1%. The day-to-day standard deviation over 5 days was SD (%) < 1%. Ceftriaxone The content of ceftriaxone was determined by an HPLC method according to the USP 23 Monograph ceftriaxone sodium. A reversed phase column (Merck AG, LiChrospher 100 RP 18, Lot 1.50828, Germany) was used with an eluent of tetraheptylammonium bromide buffer and acetonitrile, flow rate 2.0 mL/min. The samples were detected at 270 nm. The ceftriaxone content was determined after 0, 1, 2, 5, and 10 days at room temperature, as well as after 0, 1, 7, 14, 21, and 28 days under refrigeration (28C). Before each test the refrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 100300 mg ceftriaxone/L (Promochem, CRS standard ceftriaxone disodium, Lot 1, Germany). The selectivity was detected by injecting a sample of 0.9% normal saline and checking for deviations in the baseline, no deviation being observed. The reproducibility of ceftriaxone was measured using six injections. This determination gave a precision of SD (%) < 1%. The day-to-day standard deviation over 8 days was SD (%) < 1%. Ceftazidime The content of ceftazidime was determined by an HPLC method according to the USP 23 Monograph ceftazidime. A reversed phase column (Merck AG, LiChrospher 100 RP 18, Lot 1.50828, Germany) was used with an eluent of phosphate buffer (pH 7) and acetonitrile (98:2), flow rate of 2.0 mL/min. The samples were detected at 254 nm. The ceftazidime content was determined after 0, 6, 24, and 48 hours at room temperature, as well as after 0, 1, 7, 10, and 14 days under refrigeration (28C). Before each test the samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 50150 mg ceftazidime/l (Promochem, USP standard ceftazidime, Lot G, Germany). The selectivity was detected by injecting a sample of 0.9% normal saline and checking for deviations in the baseline, no deviation being observed. The reproducibility of ceftazidime was measured using six injections. This determination gave a precision of SD (%) < 1%. The day to day standard deviation over 8 days was SD (%) = 1%. RESULTS AND DISCUSSION The results of the physical investigations are presented in Table 1. The subvisible particle counts fulfil the requirements of USP 23 throughout all six studies. Changes in UV absorbance, pH and appearance are indicative of the losses in potency for all the cephalosporins studied. The concentrations of the cephalosporins were measured after fixed intervals (see Tables 2 and 3). After storage in the dark the contents of the cephalosporins were measured by multiple analysis. The samples from the Biofine containers and the control containers (glass bottles) were each determined four times at the set time points. The results are represented by average values and as percentages of the starting concentration with the t0 value being normalized to 100%. If the loss of concentration during the examination period is less than 10% the solution can be considered as stable, provided that the breakdown products are not known to be toxic. Cephalosporins differ widely in their stability in normal saline solutions (see Tables 2 and 3), and

therefore the results will be discussed individually. Cefazolin, at a concentration of 10 mg/mL, was stable in the new container for 7 days at room temperature and for 28 days under refrigeration (28C) (see Figure 3). Within these times only approximately 5% of the initial potency was lost. These results are in agreement with the literature [5], which states that the rate of degradation is temperature dependent. No significant influence of the packaging material on the stability of cefazolin in 0.9% sodium chloride was detected during the investigation. Cefotiam. The concentration, 20 mg/mL, fell substantially during the investigation period in both packaging materials. Therefore cefotiam was stable for only 12 hours at room temperature and 1 day refrigerated (see Figure 3). We have accepted 89.82% as stable, as this value, with its associated analytical error (day-to-day standard deviation: 1.3%) is to all intents and purposes 90%. The degradation rate is identical for both the new container material and the controls. Cefuroxime, at a concentration of 7.5 mg/mL, was stable in 0.9% sodium chloride for 24 hours at room temperature and for 7 days refrigerated (see Figure 3). These results correlate with the literature [6,7]. Compared with the reference standards stored in glass bottles, no significant influence of the packaging material could be detected on the stability of cefuroxime. Cefotaxime, at a concentration of 20 mg/mL, was stable in 0.9% sodium chloride solutions for 24 hours at room temperature as well as under refrigeration. The loss in potency after 4 days refrigerated was approximately 10%, in both container types (see Figure 3). These results correlate with the literature [8] where the same shelf life for cefotaxime was given. Ceftriaxone. At room temperature the concentration fell by approximately 5% within 5 days and 13% within 10 days. These results correlate with the literature [9]. In light of these results ceftriaxone was assigned a 5-day shelf-life under the conditions used. At 4C ceftriaxone was stable over the whole investigation period of 28 days, with the loss in potency being less than 5%. Ceftazidime, at a concentration of 20 mg/mL, was stable at room temperature for 48 hours and for 10 days stored in a refrigerator, in the new container as well as in the glass bottles. Similar stability data were described in the literature [10]. The loss in potency was approximately 10% over the investigation period in both container materials. Table 4 shows the inherent stability of the six cephalosporins under the conditions of the studies. With the exception of cefotiam, which has an inherently crowded hydrolysis site at C3, the remaining compounds appear to have a predictable trend. The fourth and fifth ranked compounds, cefuroxime and cefotaxime respectively, have simple ester linkages with no adjacent ring structures. Ceftazidime in third place, while hindered by the proximity of the ring, is polarized due to the positive charge present. The two most stable compounds present have a carbon sulphur linkage which gives a higher degree of hydrolytic resistance most likely due to sulphurs relatively lower electronegativity which results in less polarization of the bond. CONCLUSION The stability studies for the named cephalosporins gave shelf lives that correlate with the literature. The four graphs in Figure 3 show the decrease in concentration of the active compound, at both temperatures studied, along with the results of the glass bottle controls. Taking into account the sum of the analytical errors involved there is no significant difference between the new container made of Biofine and the glass controls. The new material does not, therefore, catalyse the hydrolysis process or display any surface phenomena.

References [1] European Pharmacopoeia Third Edition Supplement 2000, Council of Europe, Strasbourg 1999; 146. [2] DIN 58363 Infusionsbehltnisse und Zubehr, DIN Deutsches Institut fr Normung e.V., Beuth Verlag GmbH, Berlin. 1996. [3] Mc Evoy GK, editor. American hospital formulary service drug information 1999. Bethesda (American Society of Health-System Pharmacists). 1999; 12331. [4] Connors KA, Amidon GL, Stella VJ. Chemical Stability of Pharmaceuticals A Handbook for Pharmacists. 2nd Edition, 6381. [5] Ahmed I, Day P. Stability of cefazolin sodium in various artificial tear solutions and aqueous vehicles. Am J Hosp Pharm 1987; 44: 228790. [6] Faouzi MA, Dine T, Luyckx M et al. Stability and compatibility studies of cefaloridine, cefuroxime and ceftazidime with PVC infusion bags. Pharmazie 1994; 49: 4257. [7] Ahmed ST, Parkinson R. The stability of drugs in prefilled syringes: flucloxacillin, ampicillin, cefuroxime and ceftazidime. Hosp Pharm Pract 1992; 2: 2858. [8] Foley PT, Bosso JA, Bair JN, Townsend RJ. Compatibility of clindamycin phosphate with cefotaxime sodium or netilmicin sulfate in small-volume admixtures. Am J Hosp Pharm 1985; 42: 83943. [9] Kedzierewicz F, Finance C, Nicolas A, Dixneuf P, Hoffman M. Stability of parenteral ceftriaxone disodium solutions in frozen and liquid states: effect of freezing and microwave thawing. J Pharm Sci 1989; 78: 737. [10] Walker SE, Dranitsaris G. Ceftazidime stability in normal saline and dextrose 5% in water. Can J Hosp Pharm 1988; 41: 6571.

ACKNOWLEDGEMENTS The authors acknowledge the technical assistance of M Buchmller and S Sauter. Address for correspondence Dr Hans-Jrg Mller, Innovation Centre i.v. Therapies Pfingstweide 53 61169 Friedberg Germany