STABILITY STUDY OF CEFIXIME AND POTASSIUM CLAVULANATE RECONSTITUTABLE ORAL SUSPENSION BY ACCELERATED AND LONG TERM STUDIES

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STABILITY STUDY OF CEFIXIME AND POTASSIUM CLAVULANATE RECONSTITUTABLE ORAL

SUSPENSION BY ACCELERATED AND LONG TERM STUDIES

Dr. Binod Kumar Rai

¹

, Chandan Kumar

²

1Department of Chemistry, L.N.T. College, Muzaffarpur, (India)

2Department of Chemistry, B.R.A Bihar University, Muzaffarpur, (India)

ABSTRACT

Stability studies of Cefixime and potassium clavulanate reconstitutable oral suspension was performed as per the ICH guidelines Q1A (R2). The various parameters analysed include Description, pH, water content, assay and shelf life determination. These parameters were evaluated at zero month, 1st month, 2nd month, 3rd month and 6th month intervals.The conditions under which these parameters were analyzed include RT and accelerated Condition. The description of the oral suspension was evaluated as per the specifications for the time periods specified. Ithas been observed that there is significant changes in the description of the powder in accelerated condition. The pH test has been done by calibrated pH meter simultaneously during the assay analysis.The assay of cefiximeand clavulanic acid was carried out using HPLC method. The results of assay indicate that the suspension is within the allowable limits (90 - 120 %). The shelf life of the Suspension was found to be 24 months by the linear regression analysis of the stability data generated. In view of the results obtained that Cefixime and clavulanate oral suspension are recommended to be stored at 25°C and the stability of the oral suspension confirmed the ICH & USFDA guidelines.

Keywords: Cefixime, HPLC, Potassium Clavulanate, Ph, Stability Evaluation Study Etc I. INTRODUCTION

Stability testing of pharmaceutical products is a complex set of procedures involving considerable cost, time consumption and scientific expertise in order to build in quality, efficacy and safety in a drug formulation.

Scientific and commercial success of a pharmaceutical product can only be ensured with the understanding of the drug development process and the myriad tasks and milestones that are vital to a comprehensive development plan. The most important steps during the developmental stages include pharmaceutical analysis and stability studies that are required to determine and assure the identity, potency and purity of ingredients, as well as those of the formulated products (Singh et al., 2000). Stability of a pharmaceutical product may be defined as the capability of a particular formulation in a specific container/closure system to remain within its physical, chemical, microbiological, toxicological, protective and informational specifications (Kommanaboyina et al., 1999). In other words, it is the extent to which a product retains, within the specified limits, throughout its period of storage and use, the same properties and characteristics possessed at the time of its packaging.

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144 | P a g e Stability testing thus evaluates the effect of environmental factors on the quality of the a drug substance or a formulated product which is utilized for prediction of its shelf life, determine proper storage conditions and suggest labelling instructions. Moreover, the data generated during the stability testing is an important requirement for regulatory approval of any drug or formulation (Singh et al., 2000). Stability testing is termed as a complex process because of involvement of a variety of factors influencing the stability of a pharmaceutical product. These factors include stability of the active ingredient(s); interaction between active ingredients and excipients, manufacturing process followed, type of dosage form, container/closure system used for packaging and light, heat and moisture conditions encountered during shipment, storage and handling. In addition, degradation reactions like oxidation, reduction, hydrolysis or racemization, which can play vital role in stability of a pharmaceutical product, also depend on such conditions like concentration of reactants, pH, radiation, catalysts etc., as well as the raw materials used and the length of time between manufacture and usage of the product. A pharmaceutical product may undergo change in appearance, consistency, content uniformity, clarity (solution), moisture contents, particle size and shape, pH, package integrity thereby affecting its stability. Such physical changes may be because of impact, vibration, abrasion, and temperature fluctuations such as freezing, thawing or shearing etc. The chemical reactions like solvolysis, oxidation, reduction, racemization etc. that occur in the pharmaceutical products may lead to the formation of degradation product, loss of potency of active pharmaceutical ingredient (API), loss of excipient activity like antimicrobial preservative action and antioxidants etc. (Carstensen et al., 2000). Stability of a pharmaceutical product can also be affected because of microbiological changes like growth of microorganisms in non sterile products and changes in preservative efficacy (Matthews et al., 1999). The primary reason for stability testing is the concern for the well-being of the patient suffering from the disease for which the products is designed. Apart from degradation of the unstable product into toxic decomposition products, loss of activity up to a level of 85% of that claimed on the label may lead to failure of the therapy resulting in death e.g. nitroglycerine tablets for angina and cardiac arrest. Because of this concern, it has become a legal requirement to provide data for certain types of stability tests for the regulatory agencies before approval of a new product. Second important concern is to protect the reputation of the manufacturer by assuring that the product will retain fitness for use with respect to all functionally relevant attributes for as long as they are on the market. Other benefits of stability studies at the developmental stage or of the marketed products are to provide a database that may be of value in selection of adequate formulations, excipients and container closure systems for development of a new product, to determine shelf life and storage conditions for development of a new product, preparation of registration dossier, to substantiate the claimed shelf life for the registration dossier and to verify that no changes have been introduced in the formulation or manufacturing process that can adversely affect the stability of the product (Singh et al., 2000; Carstensen et al., 2000).

Table1: Stability Study Storage Conditions

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145 | P a g e Stability study Storage conditions Frequency of testing

Accelerated 40°C ± 2°C/ 75 ± 5% RH Initial, 1, 2, 3 & 6 months

Intermediate 30°C ± 2°C/ 65 ± 5% RH Initial, 3, 6, 9, 12, 18,24 & 36 months Long term/RT 25°C ± 2°C/60 ± 5% RH Initial, 3, 6, 9, 12, 18,24 & 36 months

1.1 Real-Time Stability Testing or Long Term Stability Testing

Real-time stability testing is normally performed for longer duration of the test period in order to allow significant product degradation under recommended storage conditions. The period of the test depends upon the stability of the product which should be long enough to indicate clearly that no measurable degradation occurs and must permit one to distinguish degradation from inter-assay variation. During the testing, data is collected at an appropriate frequency such that a trend analysis is able to distinguish instability from day-to-day ambiguity.

(Anderson et al., 1991).

1.2 Accelerated Stability Testing

In accelerated stability testing, a product is stressed at several high (warmer than ambient) temperatures and the amount of heat input required to cause product failure is determined. This is done to subject the product to a condition that acceleratesdegradation. This information is then projected to predict shelf life or used to compare the relative stability of alternative formulations. This usually provides an early indication of the product shelf life and thus shortening the development schedule. In addition to temperature, stress conditions applied during accelerated stability testing are moisture, light, agitation, gravity, pH and package (Kommanaboyina et al., 1999).

1.3 Experimental

1.3.1 Material and Methods

Cefixime and Potassium Clavulanate oral suspension, the working standard: working standard for cefixime and Potassium Clavulanate, Acetonitrile and Tetrabutyl ammonium Hydroxie solution (10 % aqueous solution), Orthophosphoric acid, Instruments Applied for Analytical Purpose: pH Meter, KF titrator, HPLC, Stability Chamber were obtained from A.L. Ltd. Sikkim, India.

1.3.1.1 Estimation of Cefixime and Potassium Clavulanate oral suspension by HPLC method:The assay of Cefixime and Clavulanic acid was carried out using HPLC method, which is designated as stability indicating method because of its capability to distinguish the degraded product with parent one. The following table shows the chromatographic conditions.

Table 2: Chromatographic Condition

Column 250 mm x 4.6 mm x 5 µm, ODS(preferably Supelco 516 C-18 DB)

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Flow rate 1.5 ml/min

Detector UV-VIS

Wavelength 220 nm

Injection Volume 20 µl.

Column Temperature 30ºC

1.4 Stability Study

Stability study as per the ICH guidelines was carried out for the Cefixime and Potassium clavulanate oral suspension.

Table 3: Stability Protocol and Product Details

Product Name Generic Name Label Claim

Cefixime and Clavulanate Potassium for oral suspension (50 mg/5 ml+31.25 mg/5 ml)

Cefixime and Clavulanate Potassium for oral suspension

(50 mg/5 ml+31.25 mg/5 ml)

Assay (% label claim) Each 5 ml contains:

Cefixime 50 mg

Clavulanic acid 31.25 mg Freely reconstituted

Table 3: API’s Details

Name of the API Cefixime IP as Trihydrate

equivalent to Anhydrous Cefixime Potassium Clavulanate Diluted IP equivalent to Clavulanic Acid

Primary Pack 30 ml Amber coloured Bottle with Aluminium cap.

Table 4: Packing Components Details

Primary Pack

30 ml Amber coloured Bottle with Aluminium cap.

PL. inner plug 20 mm Aluminium screw cap 20 mm

Secondary Pack

PP cap

P.L.M cup 10 ml

The sample testing intervals, number of containers to be discharged and pharmaceuticals to be analyzed in table 4 and 5.

Table 5: Storage Conditions as per ICH Guidelines Q1A (10)

Table 6: Stability Study Plan

Storage Conditions Temperature Humidity

Accelerated 40°C ± 2°C 75 ± 5% RH

Long term/RT 25°C ± 2°C 60 ± 5% RH

Sr. No. Test interval (Months) No. of Containers to Parameters to

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Table 7: Parameters to be Analyzed

Sr. No Parameter Category

1. Description I

2. pH II

3. Water Content (By KF) III

4. Assay (By HPLC) IV

Table 8: Acceptance Criteria for Parameters Tested

Sr.No. Tests Specification

1. Description (As is) Reconstituted (5.0 g /30 ml)

White to off-white granular free flowing powder filled in 30 ml amber coloured glass bottle.

White coloured homogenous suspension with flavour.

2. pH Between 3.5 and 4.5

3. Water Content (By KF) NMT 7.5% w/w 4. Assay (% label claim)

Each 5 ml contains:

Cefixime 50 mg

Clavulanic acid 31.25 mg Freely reconstituted

NLT 90.0% and NMT 120.0% of the labeled amount of Cefixime.

NLT 90.0% and NMT 120.0% of the labeled amount of Clavulanic acid.

Table 9: Evaluation of Stability Data to Establish Shelf Life - for Drug Products

If accelerated stability data for 6 months is OK.

X Y

Accelerated (6 Months)

Long term (9 months OK)

Y = 2X

Shelf life period is 18 months Accelerated

(6 Months)

Y = 2X

(6 Months)

Y = X + 12

(6 Months)

Y = X + 12

(6 Months)

Y = X

Shelf life period is 36 months Accelerated Long Term be discharged be analyzed

1 0 0 3 I,II,III and IV

2 1 - 3 I,II,III and IV

3 2 - 3 I,II,III and IV

4 3 3 3 + 3 I,II,III and IV 5 6 6 3 + 3 I,II,III and IV

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148 | P a g e If accelerated stability data for 6 months is NOT OK.

X Y

Accelerated (6 Months)

Intermediate (12 months OK)

Y = 1.5X

(6 Months)

Intermediate (9 months OK)

Y = 1.5X

Shelf life period is 13.5 months Accelerated

(6 Months)

Intermediate 9 months not OK and Long term 9 months OK

Y = X + 3

Shelf life period is 12 months

II. RESULT AND DISCUSSION

Cefixime and Potassium Clavulanate oral suspension in the final package were tested for the analytical parameters such as description, pH, Moisture content and Assay the results given in tables (10). The remaining sample containers were divided to get twice the number required to carry out the analysis at prescribed sampling intervals, to compensate any problems during the analysis.

After giving the first injection of standard solution, the chromatogram was processed and system suitability report was generated, in each case starting from zero to 6th month. It was observed that the plate count for Cefixime and clavulanic acid was found to be 23213425 and 13178785 respectively. The column efficiency was found to be more than 2500 theoretical plates for both the peaks; therefore the system was suitable and appropriate for further study, for the rest of the months. The % RSD for the peaks was found to be 0.06% and 0.07% which is with in the allowable limit of 2.0 %. The initial amount of Cefixime and clavulanic acid was found to be 54.25 mg and 36.88 mg respectively. The % potency was found to be 108.5 % and 118.0 % for cefixime and clavulanic acid respectively. It was observed that the plate count for cefixime and clavulanic acid in first month was found to be 23201239 and 13167858 respectively. For the second month, it was observed that the plate count for cefixime and Clavulanic acid was found to be 24233726 and 13485418 respectively. For the third month, it was observed that the plate count for cefixime and clavulanic acid was found to be 22533728 and 13869458 respectively. For the sixth month, it was observed that the plate count for cefixime and clavulanic acid was found to be 22321715 and 13753254 respectively. The % potency and result for both the conditions are given in table 10 & 11 respectively.

Table 10: % Assay Potency Result of Cefixime and Clavulanic acid in Accelerated Condition

Test Interval

Initial (0 month)

1^st month

2^nd month

3^rd month

6^th month

Cefixime 103.80 % 102.60 % 101.70 % 100.80 % 99.80 %

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149 | P a g e Potassium

clavulanate 102.80 % 101.70 % 100.80 % 99.70 % 98.50 %

Table 11: % Assay potency result of Cefixime and Clavulanic acid in Long term condition

Test Interval Initial

(0 month)

3^rd month

6^th month

Cefixime 103.80 % 102.00 % 101.90 %

Potassium clavulanate 102.80 % 101.00 % 100.10 %

Table 12: % Water and pH Observation at Accelerated Condition

Test Interval

1^st month

2^nd month

3^rd month

6^th month

pH 4.01 3.98 4.10 4.09 4.12

% Water 1.69 % 1.98 % 2.15 % 2.28 % 2.49 %

Table 13: % Water and pH Observation at Long Term Condition

Test

Interval

3^rd month

6^th month

pH 4.01 4.15 4.18

% Water 1.69 % 1.78 % 1.99 %

It was observed that the loss in potency of Clavulanic acid is more compared to potency of cefixime. Though there is increased loss in potency of Clavulanic acid, it is considred to be insignificant, since the loss is < 5 % (as per Q1A (R2) and USFDA) i.e from 102.8 % to 98.5% and 100.1% in accelerated and long term condition respectively.

The above all the parameter tested results showing within specification as per given in table 8. The data in table 10 and 11 was subjected to one way ANOVA – Design 1 between subject factors using ezANOVA statistical package. Pairwise comparison yielded the p<0.05 which clearly indicated not significant with very small standard deviation value.

III. CONCLUSION

Stability evaluation study of cefixime and potassium Clavulanate oral suspension was performed as per ICH guidelines Q1A(R2) by analyzing various parameters such as description,pH, water content and assay at initial, 1 month, 2 months, 3 months and 6 months interval at room temperature (25±2°C/60±5%RH) and accelerated conditions (40±2°C/75±5%RH) . All the parameters do not found to be changed significantly over a study period (6 months). The shelf life of the oral suspension was found to be 24 months by linear regression analysis of stability data generated. In view of the results obtained that cefixime and Clavulanic acid oral suspension tested are recommended to store at 25°C and the stability of oral suspension confirmed the ICH & USFDA guidelines.

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Fig: % Assay Results of Cefixime and Potassium Clavulanate at Accelerated Condition.

Fig: % Assay Results of Cefixime and Potassium Clavulanate at Long Term Condition Condition.

IV. ACKNOWLEDGEMENT

The present work on “Stability Evaluation Study of Cefixime and Potassium clavulanate reconstitutable oral suspension” is out come of Co-pilation and Computational work in Corporating my original study of the product. During my study I have received my useful suggestion and help from my Guide Dr. Binod Kumar Rai (Department of Chemistry, LNT College Muzaffarpur) is very profound scholar of inexhaustible stamina, he has given their valuable information and help during my researchwork, without all of their kind Cooperation I would not be able to complete my work.I am much thankful, especially to A.L Ltd. Sikkim, for providing the nice facility for work.

V. SOME REFERENCES OF WORK RELATED TO THE PROJECT Journals Papers

[1]. Adam, D.; Hostalek, U. and Tröster K. 5-day Cefixime therapy for bacterial pharyngitis and/or tonsillitis:

Comparison with 10-day Penicillin V therapy, Infection, (1995), 23, S83-S86.)

[2]. McMillan, A. and Young, H. The treatment of pharyngeal gonorrhoea with a single oral dose of Cefixime, International Journal of STD and AIDS, (2007), 18, 253-254.

103.80%

102.60%

101.70%

100.80% 99.80%

102.80%

101.70%

100.80%

99.70%

98.50%

95.00%

96.00%

97.00%

98.00%

99.00%

100.00%

101.00%

102.00%

103.00%

104.00%

105.00%

Initial 1 month 2 month 3 month 6 month

Cefixime Pot. Clav

103.80%

102.00% 101.90%

102.80%

101.00%

100.10%

98.00%

99.00%

100.00%

101.00%

102.00%

103.00%

104.00%

105.00%

Initial 3 month 6 month

Cefixime Pot. Clav

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151 | P a g e [3]. Accelerated Conditions of Temperature and Humidity in the Absence and the Presence of light. Part

Carstensen JT., Rhodes CT. Clin. Res. Drug Reg. Affairs.1993; 10:177-185.

[4]. Swarbrick, J, Boylan, J.C., Encyclopedia of Pharmaceutical Technology - 2nd Ed., Vol.2, [5]. Marcel Dekker Inc., New York, 577, 1211-1218, 1992.

[6]. Food and Drug Administration Guideline for submitting Documentation for the stability of [7]. Human Drugs and Biologics, U.S Department of Health and Human Services, Washington DC, [8]. 1987.

Books

[1]. Indian Pharmacopoeia 2010 , Volume II , 6th edition , 1 sep 2010, p 1012 [2]. Indian Pharmacopoeia 2010 , Volume III , 6th edition , 1 sep 2010, p 193

[3]. United States Pharmacopoeia, 30th, Rockville, MD: USP Convention Inc; 2007. p. 1654.

[4]. Guidance for Industry: Q1E Evaluation of Stability Data. U.S Department of Health and Human [5]. Services, FDA, CDER, CBER, Rockville, MD, ICH, June 2004.

[6]. ICH harmonized tripartite guideline, stability testing of new drug substances and products, Q1A (R2) Feb, 2003. p. 1-15

[7]. British Pharmacopoeia, British Pharmacopoeia Commission. Vol. 1, London: Her Majesty’s Stationary Office; 2009. p. 397.

Proceeding or Abstract

[1]. Jump up^ Joint Formulary Committee. British National Formulary, 47th edition. London: British Medical Association and Royal Pharmaceutical Society of Great Britain; 2004.

[2]. IDMA BULLETIN. Weekly publication. India drug manufacturer’s association, Bombay, [3]. India, Vol. XXVII, 21st March 1997.

[4]. Samir, A.H., Stability-Kinetics, Pharmaceutical Dosage forms. Tablets, Vol.3, Marcel Dekker Inc., New York and Basel, 339-340, 364-366, 1982.

Reports

[1]. The United States Pharmacopeia 28, The National Formulary 23, Asian edition. United States Pharmacopeial convention Inc., Rockville, MD, 143-148, 488-489, 2727-2728, 2005.

[2]. Proposed guidelines for stability studies for human drugs and biologicals, Food and Drug Administration, Washington, DC, March, 1984.

Web Sites

[1]. Prescribing information for Cefixime & Prescribing information for Clavulanic acid (www.drugs.com) [2]. http://www.fda.gov/cder/guidelines.htm

[3]. http://www.fda.gov/cber/guidelines.htm [4]. http://www.fda.gov/cber/gdlns/ichstabdta.htm