International Journal of Research in Pharmaceutical and Biomedical Sciences
ISSN: 2229-3701
______________________________________________________________________Research Paper
Development of Validated Stability Indicating RP-HPLC Method for the estimation of Capecitabine in Pure and Pharmaceutical Formulations Pani Kumar AD, Venkata Raju Y*, Sunitha G, Rama Krishna K, Ceema M and Venkateshwara Rao A Department of Pharmaceutical Analysis and Quality Assurance, Gokaraju Rangaraju College of Pharmacy, Hyderabad, AP, India __________________________________________________________________________________________ ABSTRACT A simple, rapid accurate and stability indicating RP-HPLC method was developed for the determination of Capecitabine in pure and tablet dosage form. The method showed a linear response for concentrations in the H range of 70-120 µg/ml using 0.05M phosphate buffer (p 3.0 ± 0.05) buffer and acetonitrile (50:50 % w/v) as the mobile phase with detection at 240 nm and a flow rate of 1 mL/min and retention time 4.108 min. The method was statistically validated for accuracy, precision, linearity, ruggedness, robustness, forced degradation, solution stability and selectivity. Quantitative and recovery studies of the dosage form were also carried out and analyzed; the % RSD from recovery studies was found to be less than 1. The specificity of the method was ascertained by forced degradation studies by acid, alkali hydrolysis, oxidation and thermal degradation. The degraded products were well resolved from the analyte peak with significant differences at their Rt values. Due to simplicity, rapidity and accuracy of the method, we believe that the method will be useful for routine quality control analysis. Key Words: Capecitabine, RP-HPLC, forced degradation INTRODUCTION Stability indicating methods have become an important aspect of any analytical method validation and a part of US FDA requirements. Chemically Capecitabine is designated as pentyl[1(3,4-dihydroxy-5-methyl-tetrahydrofuran-2-yl)- 5fluoro-2-oxo-1H-pyrimidin4yl]aminomethanoate1, it is used as antineoplastic agent for the treatment of breast and colorectal cancer. Literature survey reveals a few LC-MS methods reported for the determination of Capecitabine and its metabolites in biological fluids2-7, and a single HPLC method in tablet formulation7-8. Keeping this point into consideration, an attempt was made to develop a simple, accurate and validated stability indicating RP-HPLC method for the estimation of Capecitabine in pure and tablet form. The proposed method was validated as per ICH guidelines Q2A.9 MATERIALS AND METHODS Instruments Used All analytical works performed on Shimadzu HPLC-LC-20AD series binary gradient pump with _________________________________________ *Address for correspondence: E-mail:
[email protected]
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Shimadzu SPD-20A UV detector, LC-solution version software, inertsil C8 column (250 X 4.6 mm, 5 µm particle size) as stationary phase, a calibrated electronic single pan balance Shimadzu (AUX-220), a pH meter of Elico (LI-120) and ultra sonic cleaner (SONICA) were also used during the analysis.
Fig 1: Molecular structure of Capecitabine Reagents and chemicals Analytically pure Capecitabine has been obtained as a gift sample form Dr Reddy’s Laboratories Private Limited (Hyderabad, India). Tablet (Xabine) was purchased from the local market. Potassium dihydrogen orthophosphate was ortho phosphoric acid used were AR grade from Hi-media. All solvents and reagents used were of AR/HPLC grade.
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International Journal of Research in Pharmaceutical and Biomedical Sciences
ISSN: 2229-3701
Preparation of mobile phase and standard stock solution The mobile phase was prepared by mixing 500 ml of 0.05M potassium dihydrogen ortho phosphate (pH adjusted to 3.0. ± 0.05 with orthophosphoric acid) buffer with 500 ml of acetonitrile. The mobile phase was sonicated 10 min and then it was filtered through a 0.45µ membrane filter paper. An accurately weighed quantity of 50 mg was transferred to 50 ml volumetric flask, which was then dissolved and made upto volume with mobile phase to give 1000µg/ml.
Linearity The linearity range was found 80-120 µg/ml. The regression equation for Capecitabine was found to be y = 418764x + 11793 and correlation co-efficient (r2 = 0.999).
Optimized chromatographic conditions RP-HPLC analysis was performed by iso-cratic elution with flow rate of 1 mL/min. The mobile phase containing 0.05 M potassium dihydrogen ortho phosphate (pH adjusted to 3.0 ± 0.05 with ortho phosphoric acid) buffer and acetonitrile in the ratio of 50:50 (% v/v) to obtain well resolved peak of Capecitabine (Rt = 4.108 min) as shown in figure 2. wavelength of maximum absorption was selected by UV-detector. The drug shows good response at 240 nm.
Accuracy It was found out by recovery study10 using standard addition method. Known amounts of standard Capecitabine was added to pre-analyzed samples at a level from 80% upto 120% and then subjected to the proposed HPLC method. Results of recovery studies are shown in Table 4.
Calibration curve for Capecitabine Appropriate aliquots of standard stock solution were taken in different 10 ml volumetric flasks and diluted upto mark with mobile phase to obtain final concentration of 70, 80, 90, 100, 110 and 120 µg/ml of Capecitabine respectively. The solutions were injected using a 20 µg/ml fixed loop system and chromatograms were recorded. Calibration curve was drawn by plotting average peak area versus concentration as shown in figure 3. The linearity table of Capecitabine is shown in Table 1. Analysis of the marketed formulations Twenty tablets (Xabine 500 mg) were weighed accurately and crushed to form fine powder. Accurately weighed quantity of powder equivalent to about 50 mg of Capecitabine was dissolved in 50 ml of volumetric flask with mobile phase. The flask was sonicated for 20 min and then the solution was filtered using Whatmann filter paper. Appropriate volumes of the aliquot were transferred into six different 10 ml volumetric flasks and then volume was made up to the mark with mobile phase to obtain 100 µg/ml of Capecitabine. The chromatographic conditions and peak areas were measured. The results are shown in the Table 2. Validation of the method The developed method was validated in terms of linearity, accuracy, precision, specificity, limit of detection and limit of quantitation and repeatability of measurement.
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Precision Intra-day and inter-day precision of the assay samples containing Capecitabine (100 & 120 µg/ml) were analyzed six times in the same day (intra day) and for three consecutive days by different analysts. The results are shown in the Table 3.
Robustness By introducing small but deliberate changes in the mobile phase pH (± 10.0 %), mobile phase composition ( ± 2.0 % ) and flow rate (± 10.0 %) robustness of the described method were studied. The robustness of the method was assessed for 2 different amounts of calibration plot (100 & 120 µg/ml). Sensitivity The sensitivity of the method was determined with respect to LOD and LOQ. The LOD and LOQ were separately determined based on the standard calibration curve. The proposed method was shown LOD and LOQ values respectively 0.08 and 0.247. Forced degradation studies: (Specificity) The specificity of the HPLC method was determined by complete separation of capecitabine in the presence of its degradation products. The chromatogram of capecitabine along with its degradation products in 0.1 M NaOH, 0.1 M Hcl, 2% v/v H2O2. Thermal degradation at 100° C, photolytic degradation wavelength at 254 nm and 365 nm are given in figures 4, 5, 6, 7, 8 and 9 respectively. Also parameters like retention time (Rt), area and area percent also evaluated. The degradation data are shown in Table 5. RESULTS AND DISCUSSIONS Capecitabine is an oral anti neoplastic drug used for the treatment of breast and colorectal cancer. Literature survey reveals theat there is no stability indicating HPLC method reported so far for the determination of Capecitabine. Keeping this point into consideration an attempt was made to develop a simple and accurate RP-HPLC method to
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determine Capecitabine in the presence of its degradation products. The mobile phase consisting of a phosphate buffer (pH = 3.0 ± 0.05 with ortho phosphoric acid) and acetonitrile (50:50% v/v) was employed. The chromatographic separation was carried on a C8 column (Intersil) at a flow rate of 1 mL/min with UV detector at 240 nm. The run time has set at 8 min, and ambient temperatures for the HPLC system and were found to be the best for the analysis. The retention time of Capecitabine to 4.108 min with tailing factor 1.32 and number of theoretical plates 6680.35. The peak areas of the drug were reproducible as indicated by the low coefficient of variation. The amount of drug found was between 98%-102%. The sample recoveries in formulation were in good agreement with their respective label claim which suggested nointerference of formulation excipients in the estimation. Also the % RSD for both the tablet analysis and recovery studies was less than 2% indicating high degree of precision and accuracy of
ISSN: 2229-3701
the proposed method. The LOD and LOQ values are 0.08 and 0.247 µg/ml respectively. The results of the robustness study also indicated that the method is robust and is unaffected by small variations in the chromatographic conditions. Also the developed method was capable of determining Capecitabine in presence of its degradation products. Here, it can be concluded that the developed RP-HPLC method is a stability indicating simple, accurate, precise and robust method and can be employed successfully for the estimation of Capecitabine in bulk and formulation. ACKNOWLEDGEMENTS The authors are thankful to M/S Dr Reddy’s Laboratories Private Ltd, Hyderabad, India, for providing the gift sample of pure Capecitabine and for the management of Gokaraju Rangaraju College of Pharmacy for providing the essential facilities to complete this work successfully.
Table 1: Linearity of Capecitabine Concentration (µg/ml)
Area
70
3041094
80
3478922
90
3889868
100
4292618
110
4722524
120
5144837
Table 2. Analysis of commercial tablets (xabine) (n*=6) Analyte
Label claim
Amount found
(mg/tablet)
mg/tablet
500
498.55
Capecitabine
Mean
% RSD
497.05
0.20
* Mean of 6 determinations
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International Journal of Research in Pharmaceutical and Biomedical Sciences
ISSN: 2229-3701
Table 3. Precision data of the proposed method ( n* =6) Analyte
Intra day Concentration
Inter day
Area
% CV
(µg/ml) Capecitabine
Concentration
Area
% CV
(µg/ml)
100
4303066
0.024
100
4302267
0.202
120
5044562
0.032
120
5104437
0.453
* mean of six determination
Table 4 Recovery studies of Capecitabine Analyte
Capecitabine
% Type
Formulation
Amount of
Amount
% recovered
% RSD
of Level
(µg/ml)
drug added
recovered
80
50
42.01
41.84
99.6
0.35
100
50
52.47
52.57
100.3
0.26
120
50
62.54
62.98
100.7
0.25
Table 5 Forced degradation study of Capecitabine Agent
Time (hrs)
Retention Time
Area
% area
-
-
4.108
4303066
100
0.1 M NaOH
1
4.029
3611628
99.88
0.1 M Hcl
1
4.043
3313293
99.84
2% v/v H2O2
1
4.047
3700873
99.85
at 100° C
1
4.027
3611542
99.30
at UV (254 nm)
24
4.025
3665412
99.78
at UV (365 nm)
24
4.028
3634401
99.82
Fig 2: Chromatogram of standard Capecitabine
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International Journal of Research in Pharmaceutical and Biomedical Sciences
ISSN: 2229-3701
Linearity curve of Capecitabine y = 41864x + 117932 R2 = 0.9999
6000000 5000000
Calibration curve for Capecitabine
Area
4000000 3000000
Linear (Calibration curve for Capecitabine)
2000000 1000000 0 0
50
100
150
Concentration
Fig 3: Linearity curve of Capecitabine
Fig 4. Chromatogram of base (0.1 M NaOH) treated Capecitabine
Fig 5: Chromatogram of acid (0.1 M Hcl) treated Capecitabine
Fig 6: Chromatogram of Peroxides (2% v/v H2O2) treated Capecitabine
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International Journal of Research in Pharmaceutical and Biomedical Sciences
ISSN: 2229-3701
Fig 7: Chromatogram of thermal degradation (100°C) of Capecitabine
Fig 8: Chromatogram of Photolytic degradation of Capecitabine at 254 nm
Fig 9: Chromatogram of Photolytic degradation of Capecitabine at 365 nm
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