H-J Müller1, 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 NORMALSALINE SOLUTIONS, STORED IN A NEW I.V. CONTAINER MADE

OF BIOFINEÒ

The stability of six cephalosporins was studied when diluted and stored in a modified,new-generation polypropylene flexible container. Commonly used dosages of cefazolinsodium, cefotiam hydrochloride, cefuroxime sodium, cefotaxime sodium, ceftriaxonedisodium, and ceftazidime dissolved in 0.9% sodium chloride were stored both at roomtemperature and under refrigeration. The solutions were also stored in glass bottles ascontrols. Drug concentrations were measured by stability-indicating HPLC. The physicalparameters of: pH, UV absorbance, appearance, and subvisible particles were determined.Stability was defined as retention of at least 90% of the original drug concentration unlessthere were any known toxicity problems associated with the breakdown products. Theresults 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 bedetected on the stability of the investigated cephalosporins when compared with the results ofthe glass bottles. KEY WORDS: Cephalosporins, stability, compatibility, PVC-free bags

INTRODUCTIONCephalosporins are often used to treat infections with gram-negative and gram-positivemicro-organisms as well as in perioperative prophylaxis [1]. For this reason, the stability of cefazolin (afirst generation cephalosporin), cefotiam and cefuroxime (second generation cephalosporins), andcefotaxime, ceftriaxone and ceftazidime (third generation cephalosporins) were tested in the newcontainer.

Fresenius AG has developed Biofine®, a new material that combines the advantages of each availabletraditional packaging system. The product is flexible, transparent, lightweight and uses polyolefinematerials only. The primary container has been developed to meet all of the physical and mechanicalrequirements of the third edition of the European Pharmacopoeia “Plastic containers for aqueoussolutions 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, easeof recycling, and a major reduction of extractable material when compared with traditional flexiblecontainers. Figure 1 shows the chemical structure of the cephalosporins studied, with the general routes ofbreakdown 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 (2–8°C) 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 forhydrolysis; the substituted C3 position and the beta lactam ring [4]. Hydrolysis at these two sites isknown to occur either alone or in parallel depending on the pH, temperature, ionic strength, etc., of thesolvent present. Following cleavage at C3 it is usual to observe an internal rearrangement to form aninternal ester i.e. a lactone. The shelf lives obtained for the named cephalosporins, in the same infusion fluid, will primarilyrevolve around the susceptibility of the individual cephalosporins to hydrolysis. This in turn willdepend on the degree of shielding of the sites, the nature of the bond to be cleaved, the degree ofpolarization of the site and the inherent stability of the breakdown products. Under normalphysiological conditions, hydrolysis of the amide bond present does not occur. As hydrolysis is themajor 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 physicallyinteracts with the drug by mechanisms such as adsorption, absorption or permeation – phenomena notusually associated with polypropylene. Direct comparison with glass containers will identify any suchinteractions. The purpose of this study was to determine the stability of six cephalosporins when prepared in the newcontainer. This data was compared with control results generated in glass bottles. Investigation timeplans were prepared according to available literature. METHODSSample preparationUnder aseptic conditions, approximately 10 mL sodium chloride 0.9% were removed from the new

container (Fresenius AG, BiofineÒ, 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 theresultant clear solutions reinjected. Admixtures were stored at room temperature as well as in arefrigerator (2–8°C) 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

PH value Absorbance Particle Appearance

Cefazolin

(10 mg/mL)

room temperature

4°C

7 days

28 days

5.12®6.24

5.08®5.91

0.032®0.024

0.029®0.020

no increase during

investigation time

clear and

colourless

Cefotiam

(20 mg/mL)

room temperature

4°C

1 day

7 days

6.28®6.41

6.36®6.54

0.520®3.234

0.821®2.324

no increase during

investigation time

clear / light yellow

® yellow

Cefuroxime

(7.5 mg/mL)

room temperature

4°C

7 days

28 days

6.67®7.83

6.62®7.58

0.213®3.214

0.203®0.901

no increase during

investigation time

clear / light yellow

® yellow

Cefotaxime

(20 mg/mL)

room temperature

4°C

2 days

20 days

5.20®4.68

5.09®4.71

0.404®1.393

0.554®1.788

no increase during

investigation time

clear and light

yellow

Ceftriaxone

(20 mg/mL)

room temperature

4°C

14 days

28 days

6.55®7.27

6.55®6.99

0.454®3.639

0.454®2.719

no increase during

investigation time

clear / light yellow

® orange-brownish

Ceftazidime

(20 mg/mL)

room temperature

4°C

2 days

14 days

6.54®6.96

6.50®7.03

0.299®0.431

0.311®0.702

no increase during

investigation time

clear / light yellow

® yellow

Table 2. Concentration at room temperature,

Container- Investigation Time

material Initial 6 hours 12 hours 24 hours 2 days 5 days

Cefazolin

(10 mg/mL)

BiofineÒ

Glass Bottle

100%

100%

99.7%

99.6%

99.4%

99.6%

98.5%

98.6%

Cefotiam

(20 mg/mL)

BiofineÒ

Glass Bottle

100%

100%

95.8%

94.8%

89.82%

89.12%

82.3%

82.0%

Cefuroxime

(7.5 mg/mL)

BiofineÒ

Glass Bottle

100%

100%

93.4%

94.2%

86.1%

85.9%

Cefotaxime

(20 mg/mL)

BiofineÒ

Glass Bottle

100%

100%

98.2%

98.7%

92.6%

92.6%

85.0%

84.2%

Ceftriaxone

(20 mg/mL)

BiofineÒ

Glass Bottle

100%

100%

101.6%

99.0%

100.2%

98.4%

98.5%

95.3%

Ceftazidime

(20 mg/mL)

BiofineÒ

Glass Bottle

100%

100%

98.9%

98.8%

94.9%

94.2%

90.3%

90.0%

Table 3. Concentration at 2–8°C.

Container- Investigation Time

Material Initial 1 day 4 days 7 days 10 days 14 days 21 days 28 days

Cefazolin (10

mg/mL)

BiofineÒ

Glass Bottle

100%

100%

99.8%

100.0%

100.1%

100.3%

99.5%

99.4%

96.7%

96.6%

Cefotiam (20

mg/mL)

BiofineÒ

Glass Bottle

100%

100%

90.8%

91.0%

88.2%

87.8%

Cefuroxime (7.5

mg/mL)

BiofineÒ

Glass Bottle

100%

100%

96.3%

98.0%

91.1%

93.3%

84.5%

86.5%

Cefotaxime (20

mg/mL)

BiofineÒ

Glass Bottle

100%

100%

96.2%

96.4%

89.4%

90.9%

87.5%

88.3%

Ceftriaxone (20

mg/mL)

BiofineÒ

Glass Bottle

100%

100%

99.7%

100.3%

99.4%

100.7%

97.5%

99.7%

96.0%

97.4%

95.0%

97.4%

Ceftazidime (20

mg/mL)

BiofineÒ

Glass Bottle

100%

100%

100.4%

100.1%

95.2%

95.4%

92.7%

92.2%

89.2%

89.6%

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

Rank order of stability

Cephalosporin Refrigerated (2–8°C) Room temperature

Cefazolin 1 1

Ceftriaxone 2 2

Ceftazidime 3 3

Cefuroxime 4 4

Cefotaxime 5 5

Cefotiam 6 6

Physical investigationsThe determinations of the UV absorption were performed using an UV/VIS double-beamspectrophotometer (Jouan GmbH, Hitachi U‑2000, Germany), in accordance to Ph. Eur. III, 2.2.25. Thetotal spectral range (200–800 nm) was measured. If no irregular values were observed, only themeasured value at 400 nm was reported. The measurements were carried out against water as thereference 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 alight blockage particle-counter (HIAC-ROYCO, 3000A, 9064, Germany). The number of particles ³ 10µm/mL and ³ 25 µm/mL was determined. The solutions met the requirements of the test if the averagenumbers of particles present in the units tested did not exceed the limit values of 60 counts/mL ³ 10 µmand 6 counts/mL ³ 25 µm. The visual examinations were in addition to those for the subvisible particulate matter. The followingvisual changes were sought: opalescence/opacity, precipitation, gas bubble generation and anydiscoloration. Assays

The contents of cefazolin (Fresenius AG, cephazolin FreseniusÒ, Lot GI1700, Germany) cefotiam

(Takeda Pharma GmbH, SpizefÒ, Lot 897AB2, Germany), cefuroxime (Hoechst AG, ZinazefÒ, Lot

6N034, Germany), cefotaxime (Hoechst AG, ClaforanÒ, Lots N132 and N079, Germany), ceftriaxone

(Roche AG, RocephinÒ, Lots 59662 and 58262, Germany), and ceftazidime (Glaxo Wellcome,

FortumÒ, 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 fromThermo-Separation-Products (Thermo Quest, Thermo Separation Products, P4000-Pump, AS2000Autosampler, 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 outusing equipment from Hewlett Packard (Hewlett Packard, HP1100 with ChemStation, Germany) withDAD and Chem Station as control and analysis software. CefazolinThe 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 disodiumhydrogen phosphate buffer and acetonitrile (83:17), flow rate of 1.0 mL/min. The samples weredetected at 272 nm. The cefazolin content was determined after 0, 6, 24, 48 hours and after 7 days atroom temperature, as well as after 0, 1, 7, 10, and 28 days under refrigeration (2–8°C). Before each test,the refrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 50–150 mg cefazolin/L (Tiefenbacher, standard cefazolinsodium, Lot EL 1700, Germany). The selectivity was detected by injecting a sample of 0.9% normalsaline and checking for deviations in the baseline. No deviations were observed. The reproducibility ofcefazolin was measured using six injections. This determination gave a precision of SD (%) < 1%. Theday-to-day standard deviation over more than 10 days was SD (%) < 1%. CefotiamThe content of cefotiam was determined by an HPLC method in accordance with the USP 23Monograph. 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 cefotiamcontent was determined after 0, 6, 12, and 24 hours at room temperature and after 0, 1, and 4 daysunder refrigeration (2–8°C). Before each test the refrigerated samples were allowed to equilibrate toroom temperature for 6 hours. The linearity was measured over a range of 25–75 mg cefotiam/L (Grünenthal GmbH, standardcefotiam 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. Thereproducibility of cefotiam was measured using six injections. This determination gave a precision ofSD (%) < 1%. The day-to-day standard deviation over 7 days was SD (%) < 1.5%. CefuroximeThe 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 ofdipotassium hydrogen phosphate buffer and acetonitrile (83:17), flow rate of 1.0 mL/min. The sampleswere detected at 260 nm. The cefuroxime content was determined after 0, 1, and 2 days at roomtemperature, as well as after 0, 1, 7, and 14 days under refrigeration (2–8°C). Before each test therefrigerated samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 25–75 mg cefuroxime/L (Tiefenbacher, Standardcefuroxime sodium, Lot 4409100665, Germany). The selectivity was detected by injecting a sample of0.9% normal saline and checking for deviations in the baseline. No deviations were observed. Thereproducibility of cefuroxime was measured using six injections. This determination gave a precision ofSD (%) < 1%. The day-to-day standard deviation over 7 days was SD (%) < 1%. CefotaximeThe 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, Lot1.50828, Germany) was used with an eluent of buffer (containing KH2PO4, Na2HPO4, and H2O) andmethanol (83:17), flow rate of 1.0 mL/min. The samples were detected at 254 nm. The cefotaximecontent was determined after 0, 6, 24, and 48 hours at room temperature, as well as after 0, 1, 4, and 7

days under refrigeration (2–8°C). Before each test the refrigerated samples were allowed to equilibrateto room temperature for 6 hours. The linearity was measured over a range of 50–150 mg cefotaxime/L (Promochem, CRS standardcefotaxime 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. Thereproducibility of cefotaxime was measured using six injections. This determination gave a precision ofSD (%) < 1%. The day-to-day standard deviation over 5 days was SD (%) < 1%.

CeftriaxoneThe 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 rate2.0 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(2–8°C). Before each test the refrigerated samples were allowed to equilibrate to room temperature for6 hours. The linearity was measured over a range of 100–300 mg ceftriaxone/L (Promochem, CRS standardceftriaxone 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. Thereproducibility of ceftriaxone was measured using six injections. This determination gave a precision ofSD (%) < 1%. The day-to-day standard deviation over 8 days was SD (%) < 1%. CeftazidimeThe 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, and48 hours at room temperature, as well as after 0, 1, 7, 10, and 14 days under refrigeration (2–8°C).Before each test the samples were allowed to equilibrate to room temperature for 6 hours. The linearity was measured over a range of 50–150 mg ceftazidime/l (Promochem, USP standardceftazidime, Lot G, Germany). The selectivity was detected by injecting a sample of 0.9% normalsaline and checking for deviations in the baseline, no deviation being observed. The reproducibility ofceftazidime 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 DISCUSSIONThe results of the physical investigations are presented in Table 1. The subvisible particle counts fulfilthe requirements of USP 23 throughout all six studies. Changes in UV absorbance, pH and appearanceare 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 BiofineÒ containers and the control containers (glass bottles) were each determinedfour times at the set time points. The results are represented by average values and as percentages of thestarting concentration with the t0 value being normalized to 100%. If the loss of concentration duringthe examination period is less than 10% the solution can be considered as stable, provided that thebreakdown 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 roomtemperature and for 28 days under refrigeration (2–8°C) (see Figure 3). Within these times onlyapproximately 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 thepackaging material on the stability of cefazolin in 0.9% sodium chloride was detected during theinvestigation. Cefotiam. The concentration, 20 mg/mL, fell substantially during the investigation period in bothpackaging materials. Therefore cefotiam was stable for only 12 hours at room temperature and 1 dayrefrigerated (see Figure 3). We have accepted 89.82% as stable, as this value, with its associatedanalytical error (day-to-day standard deviation: 1.3%) is to all intents and purposes 90%. Thedegradation 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 roomtemperature 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 packagingmaterial 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 hoursat room temperature as well as under refrigeration. The loss in potency after 4 days refrigerated wasapproximately 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 wasassigned a 5-day shelf-life under the conditions used. At 4°C ceftriaxone was stable over the wholeinvestigation 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 10days stored in a refrigerator, in the new container as well as in the glass bottles. Similar stability datawere described in the literature [10]. The loss in potency was approximately 10% over the investigationperiod in both container materials. Table 4 shows the inherent stability of the six cephalosporins under the conditions of the studies. Withthe exception of cefotiam, which has an inherently crowded hydrolysis site at C3, the remainingcompounds appear to have a predictable trend. The fourth and fifth ranked compounds, cefuroxime andcefotaxime respectively, have simple ester linkages with no adjacent ring structures. Ceftazidime inthird 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 ofhydrolytic resistance most likely due to sulphur’s relatively lower electronegativity which results in lesspolarization of the bond. CONCLUSIONThe stability studies for the named cephalosporins gave shelf lives that correlate with the literature. Thefour graphs in Figure 3 show the decrease in concentration of the active compound, at bothtemperatures studied, along with the results of the glass bottle controls. Taking into account the sum ofthe analytical errors involved there is no significant difference between the new container made of

BiofineÒ and the glass controls. The new material does not, therefore, catalyse the hydrolysis process ordisplay any surface phenomena.

References

[1] European Pharmacopoeia Third Edition Supplement 2000, Council of Europe, Strasbourg 1999; 146.

[2] DIN 58363 Infusionsbehältnisse und Zubehör, DIN Deutsches Institut für 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; 123–31.

[4] Connors KA, Amidon GL, Stella VJ. Chemical Stability of Pharmaceuticals – A Handbook for Pharmacists. 2nd Edition, 63–81.

[5] Ahmed I, Day P. Stability of cefazolin sodium in various artificial tear solutions and aqueous vehicles. Am J Hosp Pharm 1987; 44:

2287–90.

[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: 425–7.

[7] Ahmed ST, Parkinson R. The stability of drugs in prefilled syringes: flucloxacillin, ampicillin, cefuroxime and ceftazidime. Hosp

Pharm Pract 1992; 2: 285–8.

[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: 839–43.

[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: 73–7.

[10] Walker SE, Dranitsaris G. Ceftazidime stability in normal saline and dextrose 5% in water. Can J Hosp Pharm 1988; 41: 65–71.

ACKNOWLEDGEMENTSThe authors acknowledge the technical assistance of M Buchmüller and S Sauter. Address for correspondence

Dr Hans-Jörg Müller,

Innovation Centre i.v. Therapies

Pfingstweide 53

61169 Friedberg

Germany