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STABILITY STUDY OF CEFOTAXIME SODIUM BY STABILITY INDICATING METHODS

 

Clinical courses

ABOUT AUTHORS:
Sundara Raj Behin1*, Isaac Samraj Punitha2, Sankara Subramaniyan Krishnan3
1Department of Pharmaceutics, Shree Devi college of Pharmacy, Airport Road, Kenjar, Mangalore, Karnataka – 574 142.
2Department of Pharmacognosy, Shree Devi college of Pharmacy, Airport Road, Kenjar, Mangalore, Karnataka – 574 142.
3Department of Pharmaceutics, Sri Ramakrishna Institute of Paramedical sciences, 395, Sarojini Naidu Road, Coimbatore, Tamilnadu.
*behin1@googlemail.com

ABSTRACT
Stability indicating assay methods were developed and compared for Cefotaxime Sodium. The influence of temperature and relative humidity on the stability of cefotaxime sodium in continuous infusion was investigated. The physical and chemical stability of cefotaxime sodium was determined at three different temperatures (5°C/60%RH, 25°C/60%RH, and 45°C/75%RH) and changes in the concentration of cefotaxime sodium were followed by HPTLC and microbiological assay methods. The physical stability parameters of colour change and pH were calculated. The drug solutions were clear and pale yellow initially with the intensity increasing by time, eventually becoming reddish yellow. HPTLC analysis indicated that 100mg/ml concentration of cefotaxime sodium maintained adequate stability for 2 hours at 45°C and up to 24 hours at 25°C and up to 5 days at 5°C. By microbiological assay method we found that cefotaxime sodium was stable for three days at 5ºC, one day at room temperature and four hours at 45ºC.

REFERENCE ID: PHARMATUTOR-ART-1807

INTRODUCTION
Stability testing of an active substance or finished product gives evidence about the quality of the active substance or finished product which varies over time because of the influence of environmental factors like temperature, humidity and light.  Stability testing also provides vital informations about the interaction of drug with its ingredients, possible degradations and their mechanisms, and degraded products.  The results of stability studies are widely used by the pharmaceutical industry in deciding the storage conditions, suitable packaging material, shelf life and expiration date of the product (1, 2, 3).


Stability is defined as the capacity of a drug substance or drug product to remain within established specifications to maintain its identity, strength, quality, and purity throughout the retest or expiration dating periods (4). Drug stability is aimed at ensuring that the drug product remains within specifications established to ensure its identity, strength, quality and purity. Drug stability and compatibility are critical issues controlling accurate and appropriate delivery of drug therapy to patients.  Impurities and degradation can lead to change in the pharmacological, chemical and toxicological properties of the drug which will affect its safety and efficacy (5, 6, 7). Stability is very important for antibacterial agents especially those given by Intra venous (I .V route) as they reach systemic circulation directly, and the clinical outcome and safety are directly correlated to drug levels in blood.

The increased use of parenteral drugs is revealed in surveys that show in the average hospital, 40% of the total dosage forms dispensed to patients are in the form of injections (8). Newer generation of parenteral antibioticshave lead to increased role of parenteral therapy. Continuous infusion is an efficient means of administering beta-lactams to maintain drug concentrations higher than the Minimum Inhibitory concentration (MIC) throughout the dosing interval.  Continuous infusion has a pharmacoeconomic advantage over intermittent dosing by achieving the same effect with a lower daily dose of drug.


Cefotaxime sodium is a semisynthetic, broad spectrum cephalosporin antibiotic for parenteral administration. Cefotaxime is effective in treating patients with complicated urinary tract and lower respiratory tract infections (9,10).  Most monograph literatures on cefotaxime indicate stability of the drug at room temperature (25ºC) and refrigeration (5ºC) (11,9,12).  Hence we were interested in studying if cefotaxime which is extensively used intravenously maintains its stability when used clinically as continuous infusion, in a temperate country like India, where temperature may reach up to 45oC in summer. We have also developed two stability indicating assay methods to analyse the change in concentration of the samples.

MATERIALS AND METHODS

Materials:
Cefotaxime sodium was gifted by Orchid pharma, Chennai.  Cefotaxime sodium vials were procured from Alkem Chemicals, Mumbai.  Water for injection was purchased from Core health care, Mumbai. Mueller Hinton Agar media was purchased from Hi- Media, Mumbai. All other chemicals and reagents used were of laboratory or analytical grade.

Chromatographic Conditions:
Chromatography was performed on HPTLC plates (10 ? 10 cm) (Camag) chromatographic system by the Ascending chromatography method. Compounds of interest were separated, employing ethylacetate: acetone: water: acetic acid (10:5:3:2 v/v) as mobile phase and Silicagel 60F254 as the stationary phase.  During the investigation, the mobile phase composition remained unaltered. The samples were injected at a quantity of 10mlusing Linomat injector. The peak areas were measured at 254 nm using UV detector and scanned (Camag scanner) (13).

Standard Stock solution:
1gm of cefotaxime sodium was dissolved in 10 ml of sterile water to produce 100mg/ml and serially diluted to produce 200µg/ml and final dilutions were made to the concentrations ranging 0.2 – 1 µg/spot respectively.

Standardization of Cefotaxime sodium:
Cefotaxime sodium corresponding to graded concentrations of 0.2 - 1µg/spot were spotted in HPTLC plates of size 10 × 10 cm.  The spots were dried and developed using ethylacetate: acetone: water: acetic acid (10:5:3:2) as solvent system (13) and peak areas were measured at 254nm.

Stability studies by HPTLC (14, 15):
Three 1gm vials of Cefotaxime sodium I .V were reconstituted with 10ml of sterile water for injection and were stored at 5ºC, 25ºC and 45ºC. From the above reconstituted solutions 0.2 ml solution was withdrawn and made up to 10ml and further 1ml was withdrawn and made up to 10ml and serially diluted to produce 200µg/ml.  From the above solution 0.5ml of drug solution was withdrawn and made up to 1ml with sterile water, 10µl of drug solution was spotted in HPTLC plates of size 10 × 10 cm and developed in a solvent system comprising ethyl acetate: acetone: water: acetic acid (10:5:3:2), dried and peak areas were measured at 254nm.  The above procedure was repeated 5 days with samples withdrawn from vials stored at 5ºC, 25ºC, 45ºC at various time intervals of 0min, 1hr, 2hrs, 4hrs, 7hrs, 24 hrs, 72 hrs, 120 hrs. pH and clarity were noted.  The results obtained were observed and recorded.

Stability Studies by Microbiological Assay:
Three 1gm vials of cefotaxime sodium were reconstituted with 10ml of sterile water for injection; these vials were marked as room temperature (25ºC), 45ºC and refrigeration (5ºC) for identification and were kept at different storage conditions. From the above reconstituted solutions, 0.2ml equivalent to 20mgs was pipetted and made up to 10 ml in a standard flask to a concentration of 2mg/ml.  From this solution 10µl was pipetted out to contain 20mcg of cefotaxime sodium, and was added to the sterile disc kept on Muller Hinton agar plates, already swabbed using Staphylococcus aureus as test organism.  The plates were incubated at 37ºC for 24 hours and observed for the zone of inhibition.  The above procedure was repeated for various temperatures with sampling at different time intervals up to 72 hours (0, 0.5hr, 1hr, 2hr, 3hr, 4hr, 6hr, 24hr, 48hr and 72hr).

RESULTS

Stability studies by HPTLC:
Stability study was conducted on pure drug of the investigated compound (Cefotaxime sodium). The mobile phase was selected with reference to (13) and preliminary experiments showed the elution of cefotaxime sodium in the solvent system.  Cefotaxime sodium I.V. infusion (100mg/ml) was stored at different temperatures such as room temperature (25ºC), refrigeration (5ºC) and (45ºC).  Samples were withdrawn at different time intervals for 5 days and quantitated by HPTLC method.  The stability of cefotaxime sodium I.V. infusion stored at refrigeration temperature proved to be more stable even after 72 hours (% deviation = 5.05) than the other solutions stored at room temperature (% deviation = 11.11) and 45º (% deviation = 27.83) (Table I) (Figure I).

Table: I

Chemical Stability of Cefotaxime Sodium I.V. at Three Different Temperatures

Temp

Expected

concentration

0

hour

1

hour

2

hour

4

hour

7

hour

24

hour

72

hour

120

hour

5ºC

1µg/spot

5220.7

(0.99)

5156.3

(0.98)

5145.2

(0.97)

5086.4

(0.95)

5064.7

(0.95)

5011.8

(0.94)

5003.6

(0.94)

4936.8

(0.91)

25ºC

1µg/spot

5220.7

(0.99)

5053.6

(0.96)

5041.7

(0.96)

4982.3

(0.94)

4933.7

(0.93)

4812.6

(0.91)

4654.9

(0.88)

4423.3

(0.82)

45ºC

1µg/spot

5220.7

(0.99)

5032.2

(0.94)

4822.6

(0.91)

4573.9

(0.87)

4324.3

(0.82)

3756.2

(0.71)

-

-

Values recorded are the peak area values and the values given within brackets are the concentration of cefotaxime sodium I.V. in mcg/ml.

Percentage deviation  =    Initial concentration – Final   × 100
                                             -------------------------------
                                                     Initial Concentration

At 45ºC, the % deviation was > 10% (considered unstable) after nearly 2 hours of storage (value indicates between 2-4 hours).  The degradation was also confirmed by the physical color change from colorless to pale yellow at the 2 hours sample (Table II).

Table: II

Physical Stability of Cefotaxime Sodium I.V. at Three Different Temperatures:

Temperature

 

0

hour

1

hour

2

hour

4

hour

7

hour

24

hour

72

hour

120

hour

 

5ºC

pH

5.9

5.9

5.9

5.9

5.9

5.8

5.8

5.4

Clarity

clear

clear

clear

clear

clear

clear

clear

Clear

Color

Color

less

Color

less

Color

less

Color

less

Color

less

Color

less

Color

less

Color

less

 

25ºC

pH

5.9

5.9

5.9

5.9

5.7

5.4

5.4

5.2

Clarity

clear

clear

clear

clear

clear

clear

clear

clear

Color

Color

Color

less

Color

less

Color

less

Color

less

Dark yellow

Dark yellow

Reddish

yellow

 

45ºC

pH

5.9

5.9

5.8

5.8

5.6

5.2

-

-

Clarity

clear

clear

clear

clear

clear

clear

-

-

Color

Color

less

Color

less

Pale yellow

Pale yellow

Dark yellow

Reddish yellow

-

-

At 25ºC > 10% degradation was seen between 24 hours and 48 hours.  The physical color change was seen at 24 hour sample from colorless to dark yellow and at the end of 5 days the sample was reddish yellow in color indicating complete degradation (Table III).

Table: III

Degradation Profile of Cefotaxime Sodium at Three Different Temperatures

Sampling Time

Amount of Drug Remaining (%)

5ºC

25ºC

45ºC

0 time

100

100

100

1 hour

98.99

96.97

94.95

2 hour

97.98

96.97

91.92

4 hour

95.96

94.95

87.88

7 hour

95.96

93.94

82.83

24 hour

94.95

91.92

71.72

72 hour

94.95

88.89

-

120 hour

91.92

82.83

-

At 5ºC drug solution was stable up to 5 days. There was no color change in the solution at the end of 5 days, indicating the sample was stable (Table II).  The results obtained from pH, color and clarity test showed physical stability at 5ºC even after 5 days (Table II).

Figure I: Degradation of Cefotaxime Sodium at different temperatures (5?C, 25?C and 45?C).  Samples at 45?C showing degradation at shorter period of time.

Stability Studies by Microbiological Assay:
Cefotaxime sodium was more stable at refrigeration temperature when compared with 25ºC and 45ºC as per microbiological method of assay.

Figure II
Zone of Inhibition of Cefotaxime Sodium I.V by Microbiological Assay at Different Temperatures Using Staphylococcus Aureus- Ncim 2079 as Test Organism.

As per this method cefotaxime sodium was stable at 5ºC for 72 hours, at 25ºC for 24hours and at 45ºC only for 4 hours (Table IV) (Figure II).

Table IV
Zone of Inhibition and Concentrations (With Percentage Deviation) of Cefotaxime Sodium by Microbial Assay Using Staphylococcus Aureus Ncim 2079 as Test Organism

Time of

sampling

Expected

concentration

Refrigeration

(5ºC)

Room

Temp

(25ºC)

(45ºC)

Percentage deviation

 

5oC

25oC

45oC

0

20µg/10µl

31 (20)

31 (20)

31 (20)

0

0

0

0.5 hr

20µg/10µl

31 (20)

31 (20)

31 (20)

0

0

0

1 hr

20µg/10µl

31 (20)

31 (20)

31 (20)

0

0

0

2 hr

20µg/10µl

31 (20)

31 (20)

31 (20)

0

0

0

3 hr

20µg/10µl

31 (20)

31 (20)

31 (20)

0

0

0

4 hr

20µg/10µl

31 (20)

31 (20)

31 (20)

0

0

0

6 hr

20µg/10µl

31 (20)

31 (20)

29 (10)

0

0

50

24 hr

20µg/10µl

31 (20)

31 (20)

29 (10)

0

0

50

48 hr

20µg/10µl

31 (20)

29 (10)

23 (8)

0

50

60

72 hr

20µg/10µl

31 (20)

29 (10)

21 (5)

0

50

75

Values recorded are zone of inhibition values and those given in brackets are concentration of cefotaxime sodium I.V. in mcg/spot.

DISCUSSION
Results of our study involving cefotaxime sodium (by HPTLC method) were found similar to those reported by Fabre and Eddine (14); where cefotaxime sodium was stable up to 5 days at refrigeration  temperature (5ºC) and 24 hours at room temperature (25ºC), our results indicate cefotaxime sodium was stable only for 2 hours at 45ºC.

There was no visible change in the HPTLC chromatogram of cefotaxime during our entire study excepting in the case of 45°C exposure of cefotaxime after 72hrs where no recordable peak corresponding to cefotaxime was found indicating complete degradation of cefotaxime.  Cefotaxime Sodium infusion was stable at the end of 5 days when stored at 5?C, retaining its color and pH. Hence our datas agree with the datas of the marketed product Taxim (10). When stored at 25?C, the degradation started around 24 hours, indicating stability for a day. At the accelerated stability temperature of 45?C, the degradation started around 2 hours itself.