ISSN 2230 DEVELOPMENT AND VALIDATION OF STABILITY ... · DEVELOPMENT AND VALIDATION OF STABILITY INDICATING ... It showed that there was no change in physical parameter for ... Table

Tamboli Ashpak Mubarak et al. IRJP 2 (8) 2011 185-191

IRJP 2 (8) August 2011 Page 185-191

INTERNATIONAL RESEARCH JOURNAL OF PHARMACY ISSN 2230 – 8407 Available online http://www.irjponline.com Research Article

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPLC METHOD

FOR SIMULTANEOUS DETERMINATION OF AMLODIPINE BESYLATE AND ENALAPRIL MALEATE

Tamboli Ashpak Mubarak*, Khan Naziya Iqbal, Manure Javed Yakub, Shaikh Sohrab Akhtar, Mohite Shrinivas Krishna

Department of Pharmaceutical Chemistry, Sahaydri College of Pharmacy, Methwade, Sangola (Maharashtra), India

Article Received on: 18/06/11 Revised on: 14/07/11 Approved for publication: 10/08/11 *E-mail: [email protected] ABSTRACT Stability testing is a routine procedure performed on drug substances and drug products to provide evidence on how the quality of a drug varies with time under the influence of a variety of parameters. Environmental factors such as temperature, humidity and light can potentially affect product quality and requires establishing a re-test period for the drug substance or a shelf-life for the drug product and recommended storage conditions. Companies rely on the stability data to establish an expiry for marketed pharmaceutical products. To investigate potential stability issues of the drug in AMECA tablet, accelerated and intermediated stability studies were carried out according to ICH guidelines at 300C+20C/65%RH+ 5%RH and 400C+20C/75%RH+5%RH for six month. Stability studies were carried out using programmable environmental test chamber CHM-10s, serial no. ICH-2530 and ICH 2531 (Remi instrument Ltd, Mumbai) and assay was carried out by using HPLC (Jasco) method which has been developed previously. The stability testing of AMECA tablets showed there was no significance change in the content of Amlodipine Besylate and Enalapril Maleate after 6 month of storage under controlled of temperature and humidity, at 300C+20C/65%RH+5%RH. Accelerated conditions under 400C+20C/75%RH+5%RH, dosage forms exhibit slight but insignificant fall within assay value. The parameters such as appearance, hardness, friability, disintegration time periods were determine at regular interval of each month for total period of six months. It showed that there was no change in physical parameter for six months at stored condition. KEYWORDS Amlodipine Besylate, Enalapril Maleate, HPLC, Stability study, accelerated stability studies. INTRODUCTION Stability study 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. Physical, chemical, and microbiological data are generated as a function of time and storage conditions [e.g. temperature and relative humidity (RH)]. Stability testing is the primary tool used to assess expiration dating and storage conditions for pharmaceutical products. Many protocols have been used for stability testing, but most in the industry are now standardizing on the recommendations of the International Conference on Harmonization (ICH). These guidelines were developed as a cooperative effort between regulatory agencies and industry officials from Europe, Japan, and the United States1,2. Stability is a critical quality attribute of all pharmaceutical products and therefore stability testing is a crucial component of drug development process. Companies rely on the stability data to establish an

expiry for marketed pharmaceutical products3. Many guidelines have been developed around this arena; however, many issues are continually raised and challenge our practices. Recently, the U.S. Food and Drug Administration withdrew their Stability Guidance; and ICH withdrew Q1F guidelines on storage requirements for Zone III and IV, leading manufacturers to search for the right choice of stability storage conditions for global submission4,5. MATERIALS & METHODS General information about tablet Each uncoated tablet contains Ø Amlodipine Besylate 5mg Ø Enalapril Maleate 5mg Colour: Sunset yellow FCF Storage: Storage in cool, dry and dark place. Manufactured by: Systopic Ltd. Stability Study of Amlodipine Besylate and Enalapril Maleate Tablet as per ICH Guideline To carry out the intermediate and accelerated stability studies of Amlodipine Besylate and Enalapril Maleate tablet, 500 tablets procured from market.



25 strips were stored in each programmable environmental test chamber CHM-10S, (Remi sales and engineering Ltd) calibrated previously and labeled as 300 + 20C/65% RH + 5% RH (serial no.ICH – 2530) 400 + 20C/75% RH + 5% RH (serial no.ICH – 2531) Testing of tablet was done at 0th month before keeping the strips for both intermediate accelerated stability. As per ICH guidelines further testing was done at the end of each month for 6th month6,7,8. Determination of Amlodipine Besylate and Enalapril Maleate by HPLC method Procedure- 10 Tablet each containing Amlodipine Besylate 5 mg and Enalapril Maleate 5 mg were weighed and crushed to fine power and quantity of powder equivalent to 5 mg of Amlodipine Besylate and 5 mg of Enalapril Maleate were weighed and transfer to 50 ml volumetric flask mobile phase was added to same flask and sonicated for 20 min . The volume was made up to 50 ml with mobile phase the concentration was 100 µg/ml of Amlodipine Maleate and 100 µg/ml of Enalapril Maleate. The final solution containing 100 µg/ml of Amlodipine Maleate and 100 µg/ml of Enalapril Maleate were filtered by 0.45 µm nylon membrane filters. From this solution appropriate dilution were made as to obtain 40 µg/ml of Amlodipine Besylate and 40 µg/ml of Enalapril Maleate as final concentration an injected in to system to get the chromatogram the area obtained in each chromatogram of five replicates was correlated with regression equation and amount found is calculated which was within the limits of label claimed as mention in table. RESULT AND DISCUSSION 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. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a re-test period for the drug substance or a shelf life for the drug product and recommended storage conditions. Stability data for the drug substance are used to determine optimal storage and packaging conditions for bulk lots of the material. The stability studies for the drug product are designed to determine the expiration date (or shelf life). In order to assess stability, the appropriate physical, chemical, biological and microbiological testing must be performed. Usually this testing is a subset of the release testing.

To investigate potential stability issues of the drug in AMECA tablet, accelerated and intermediated stability studies were carried out according to ICH guidelines at 300C+20C/65%RH+ 5%RH and 400C+20C/75%RH+5%RH for six month. Stability studies were carried out using programmable environmental test chamber CHM-10s, serial no. ICH-2530 and ICH 2531 (Remi instrument Ltd, Mumbai) and assay was carried out by using HPLC (Jasco) method which has been developed previously. The stability testing of AMECA tablets showed there was no significance change in the content of Amlodipine Besylate and Enalapril Maleate after 6 month of storage under controlled of temperature and humidity, at 300C+20C/65%RH+5%RH. Accelerated conditions under 400C+20C/75%RH+5%RH, dosage forms exhibit slight but insignificant fall within assay value. The parameters such as appearance, hardness, friability, disintegration time periods were determine at regular interval of each month for total period of six months. It showed that there was no change in physical parameter for six months at stored condition. Periods for electrical cut off and other related problems were compensated by extending the study for the relevant periods. ACKNOWLEDGEMENT I would like to thank Amatas Ltd., Mumbai, for providing Enalapril Maleate and Smurthi Organics Ltd. for providing Amlodipine Besylate as gift samples for my work. I am also greatly thankful to my guide Mr. S.K Mohite Sir for his encouragement and support during my work. I would also like to thank my friends for the emmense support at each and every step of the project. REFERENCES 1. General Notices- 26, “Storage, Temperature, and Humidity,” USP 27–NF 22 U.S. Pharmacopeial Convention, Rockville, MD, 2004: 10. 2. General Information- 1151, “Pharmaceutical Dosage Form,” USP 27–NF 22 U.S. Pharmacopeial Convention, Rockville, MD, 2004: 2577–2578. 3. Leon Lachman, Herbert A. Liebermann, Joseph L. Kanig; The Theory and Practices of Industrial Pharmacy, 3rd edition; 760-782. 4. Michel E. Aulton; Pharmaceutics, The science of dosage form design, 2002; 109-112. 5. Subramanyam, C.V.S.; Textbook of Physical Pharmaceutics, 2nd edition, Vallabh Prakashan, 2004: 51-77. 6. Haynes J. D; Worldwide virtual temperatures for product stability testing. Journal of Pharmaceutical Sciences, 1971, 5th edition, 2001: 927-929. 7. Schumacher P., Uber eine für die Haltbarkeit von Arzneimitteln magebliche Klimaeinteilung; The impact of climate classification on the stability of medicines; Pharm Ind 2000: 481-483. 8. Zahn M. et al. A risk-based approach to establish stability testing conditions for tropical countries, Journal of Pharmaceutical Sciences; 2006, 95: 946-965.



Table 1 Optimized HPLC Method parameter for Amlodipine Besylate and Enalapril Maleate.

Chromatographic mode

Chromatographic conditions

HPLC system

JASCO PU – 2080 plus

Pump

JASCO PU – 2080 plus Intelligent HPLC pump

Detector

UV- 2075 plus Intelligent UV/VIS detector

Stationary phase

HiQ sil C-8 (4.6* 250)

Mobile phase

0.1% orthophosphoric acid (pH- 2.8) :Acetonitrile (30:70)

Concentration of Standard solution

Amlodipine Besylate 40 micro/ml and Enalapril Maleate 40 micro/ml

Detection wavelength

232 nm

Flow rate

1 ml/min

Sample size

20 micro liter

Column temperature

Ambient

Table 2 Physical tests performed on AMACE stored at 300 + 20C/65% RH + 5%

RH Month Thickness

(mm) Hardness (kg/cm)

Friability (%)

Disintegration time

Weight Variation

(%) 0th

month 3 2.13 Complies 2.31 3.4

1st month

3 2.12 Complies 2.33 3.9

2nd month

3 2.12 Complies 2.39 3.8

3rd Month

3 2.11 Complies 2.31 3.4

4th


5th month

3 2.11 Complies 2.41 3.7

6th month

3 2.11 Complies 2.36 3.4

Table 3 Physical tests performed on AMACE stored at 400 + 20C/75% RH + 5%

RH Month Thickness

(mm) Hardness (kg/cm)

Friability (%)

Disintegration time

Weight Variation

(%) 0th


1st month

3 2.13 Complies 2.30 3.4

2nd month

3 2.12 Complies 2.45 3.4

3rd month

3 2.12 Complies 2.20 3.6

4th


5th month

3 2.11 Complies 2.41 3.9

6th month

3 2.11 Complies 2.31 3.1

Table 4 Details of chromatograms at zero month 300 C /65% RH

Drug Retention time

(min) Area (µV.sec) Assay

(%) Standard Sample Standard Sample

AMLO 3.653 3.653 5871773.06 5866609.53 100.71 ENA 2.526 2.526 4756976.70 4870370.67 100.40

Table 5 Details of chromatograms at first month 300 C /65% RH

Drug Retention time



AMLO 3.653 3.684 5877834.36 5887733.37 100.69 ENA 2.526 2.529 4763875.33 4772673.43 100.38

Table 6 Details of chromatograms at second month 300 C /65% RH

Drug

Retention time (min)

Area (µV.sec) Assay (%)

Standard Sample Standard Sample AMLO 3.659 3.653 5837539.88 58434336.78 100.60

ENA 2.536 2.526 4755336.39 4767256.23 100.31

Table 7 Details of chromatograms at third month 300 C /65% RH

Drug Retention time


(%)

Standard

Sample

Standard Sample

AMLO

3.654 3.633 5863643.79

5872539.83

100.20

ENA 2.534 2.532 4763256.33

4771451.47

100.30

Table 8 Details of chromatograms at fourth month 300 C /65% RH

Drug

Retention time (min)

Area (µV.sec) Assay (%)


ENA 2.586 2.526 4725637.47 4726537.36 100.25

Table 9 Details of chromatograms at fifth month 300 C /65% RH

Drug Retention time


(%)


ENA 2.529 2.469 4745688.29 4743588.30 100.20

Table 10 Details of chromatograms at sixth month 300 C /65% RH

Drug Retention time


(%)


ENA 2.469 2.426 4738577.19 4738492.22 100.10

Table 11 Details of chromatograms at zero month 400C /75% RH

Drug Retention time


(%)


ENA 2.536 2.536 4753779.33 4759539.43 100.55

Table 12 Details of chromatograms at first month 400C /75% RH

Drug Retention time


(%)


ENA 2.569 2.539 4743536.41 4753887.39 100.40

Table 13 Details of chromatograms at second month 400C /75% RH

Drug Retention time


(%)


ENA 2.529 2.469 4751187.39 4752198.43 99.44

Table 14 Details of chromatograms at third month 400C /75% RH

Drug Retention time


(%)


ENA 2.578 2.469 4739252.43 4740536.19 99.01



Table 15 Details of chromatograms at fourth month 400 /75% RH

Drug Retention time

(min) Area (µV.sec) Assay (%)


ENA 2.569 2.426 4729736.51 4725537.87 98.12

Table 16 Details of chromatograms at fifth month 400C /75% RH

Drug Retention time


(%)


ENA 2.532 2.413 4778903.01 4669833.03 97.02

Table 17 Details of chromatograms at sixth month 400C /75% RH

Drug Retention time



AMLO 3.633 3.426 5723305.39 5613953.44 97.10 ENA 2.532 2.402 4750309.49 4643339.38 95.32

Table 18 Assay of AMACE stored at 300 C and 65% RH by HPLC Month Amount

present (mg) Amount found

(mg) Assay (%)

AMLO ENA AMLO ENA AMLO ENA

0th Month

5 5 5.035 5.020 100.71 100.40

1st Month

5 5 5.034 5.019 100.69 100.38

2nd

Month 5 5 5.030 5.015 100.60 100.31

3rd Month

5 5 5.010 5.014 100.20 100.30

4th Month

5 5 5.005 5.012 100.10 100.25

5th Month

5 5 5.004 5.010 100.09 100.20

6th Month

5 5 5.00 5.005 100.00 100.10

Table 19 Assay of AMACE stored at 400 C and 75% RH by HPLC Month Amount

present (mg) Amount found

(mg) Assay (%)

AMLO ENA AMLO ENA AMLO ENA 0th

Month 5 5 5.010 5.027 100.20 100.55

1st Month

5 5 5.007 5.020 100.15 100.40

2nd

Month 5 5 4.968 4.972 99.36 99.44

3rd Month

5 5 4.960 4.9505 99.20 99.01

4th Month

5 5 4.933 4.906 98.67 98.12

5th Month

5 5 4.872 4.851 97.44 97.02

6th Month

5 5 4.855 4.766 97.10 95.32

ASSAY OF AMACE STORED AT RH BY USING JASCO PU 2080 HPLC SYSTEM 300 + 20C/65% RH + 5% RH

Fig 1 Chromatogram at zero month 300C /65% RH (Standard)

Fig 2 Chromatogram at zero month 300 C /65% RH (Sample)

Fig 3 Chromatogram at first month 300 C /65% RH (Standard)

Fig 4 Chromatogram at first month 300 C /65% RH (Sample)

Fig 5 Chromatogram at second month 300 C /65% RH (Standard)



Fig 6 Chromatogram at second month 300 C /65% RH (Sample)

Fig 7 Chromatogram at third month 300 C /65% RH (Standard)

Fig 8 Chromatogram at third month 300 C /65% RH (Sample)

Fig 9 Chromatogram at fourth month 300 C /65% RH (Standard)

Fig 10 Chromatogram at fourth month 300 C /65% RH (Sample)

Fig 11 Chromatogram at fifth month 300 C /65% RH (Standard)

Fig 12 Chromatogram at fifth month 300 C /65% RH (Sample)

Fig 13 Chromatogram at sixth month 300 C /65% RH (Standard)

Fig 14 Chromatogram at sixth month 300 C /65% RH (Sample)

ASSAY OF AMACE STORED AT 400C AND 75% RH BY USING JASCO PU

2080 HPLC SYSTEM



Fig 15 Chromatogram at zero month 400C /75% RH (Standard)

Fig 16 Chromatogram at zero month 400C /75% RH (Sample)

Fig 17 Chromatogram at first month 400C /75% RH (Standard)

Fig 18 Chromatogram at first month 400C /75% RH (Sample)

Fig 19 Chromatogram at second month 400C /75% RH (Standard)

Fig 20 Chromatogram at second month 400C /75% RH (Sample)

Fig 21 Chromatogram at third month 400C /75% RH (Standard)

Fig 22 Chromatogram at third month 400C /75% RH (Sample)

Fig 23 Chromatogram at fourth month 400C /75% RH (Standard)

Fig 24 Chromatogram at fourth month 400C /75% RH (Sample)



Fig 25 Chromatogram at fifth month 400C /75% RH (Standard)

Fig 26 Chromatogram at fifth month 400C /75% RH (Sample)

Fig 27 Chromatogram at sixth month 400C /75% RH (Standard)

Fig 28 Chromatogram at sixth month 400C /75% RH (Sample)

GRAPHICAL REPRESENTATION OF ASSAY RESULT

Fig 29 Assay of AMACE stored at 300 C and 65% RH by HPLC

Fig 30 Assay of AMACE stored at 400 C and 75% RH by HPLC

Source of support: Nil, Conflict of interest: None Declared

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ISSN 2230 DEVELOPMENT AND VALIDATION OF STABILITY ... · DEVELOPMENT AND VALIDATION OF STABILITY INDICATING ... It showed that there was no change in physical parameter for ... Table