VALIDATED STABILITY INDICATING HPLC METHOD FOR ESTIMATION OF CAPECITABINE

VALIDATED STABILITY INDICATING HPLC METHOD FOR ESTIMATION OF CAPECITABINE

K. V. Lalitha ^{* 1}, J. Raveendra Reddy ¹ and N. Devanna ²

Division of Pharmaceutical Analysis and Quality Assurance ¹, Raghavendra Institute of Pharmaceutical Education and Research (RIPER) - Autonomous Ananthapuramu - 515172, Andhra Pradesh, India.

Jawaharlal Nehru Technological University - OTPRI ², Ananthapuramu - 515002, Andhra Pradesh, India.

ABSTRACT: A new stability-indicating high-performance liquid chromatographic method of analysis of capecitabine in pharmaceutical dosage form was developed and validated. The solvent system consisted of methanol: water (60:40% v/v). The retention time of capecitabine was 7.927 min at a flow rate of 1 ml/min on C-18 (Quails BDS, 250 × 4.6 mm, 5µ). Capecitabine was detected at 240 nm. The linear regression analysis data for the linearity plot showed good linear relationship with the correlation coefficient value, R² = 0.9991 in the concentration range 4-50 µg/ml. The method was validated according to the ICH guidelines. The accuracy of the method was validated by recovery studies and was found to be significant and under specification limits, with Recovery 98.12-101.61%. The assay of capecitabine was determined in tablet dosage form. The method was also found to be robust. The drug was subjected to stress conditions of acidic, basic, oxidation, photolytic and thermal degradation, and considerable degradation was found in all stress conditions. The method has proven specificity for the stability-indicating method.

Capecitabine, HPLC, Degradation, Stability studies

INTRODUCTION: Capecitabine is an antineoplastic carbamate of fluoropyrimidine, and it is used in the category of anti-metabolites medicinal drugs. Capecitabine is a chemotherapy agent orally used to treat metastatic and colorectal cancers. Capecitabine is a 5'-deoxy-5-fluorouridine (5'-DFUR) drug which inhibits the synthesis of DNA and slowns the growth of tumor-tissue in cells.

This induces the pharmacodynamic response by representing a natural nutrient in cells necessary for the growth of cancer cells. Capecitabine is taken up by the cancer cells and then impairs their growth ^1-4. The literature review indicates that few experi-mental approaches have been used to measure capecitabine by HPLC ^5-8. Table 1 shows the list of formulations of capecitabine available in market.

Drug Profile of Capecitabine: ⁹

DRUG PROFILE OF CAPECITABINE

TABLE 1: LIST OF FORMULATIONS OF CAPECITABINE AVAILABLE IN MARKET

EXPERIMENTAL:

Chemicals and Reagents: HPLC grade methanol and water were purchased from Merck Company, Mumbai, India.

Method Development:

Preparation of Standard Drug Solutions: Stock solution of Capecitabine was prepared by dissolving 10 mg of Capecitabine in separate 10 mL of the volumetric flask with a small quantity of water. The mixture was sonicated for about 10 min and then made up to volume with water. From the stock solution, 100 μg/mL of capecitabine was prepared.

Chromatographic Conditions: The mobile phase consisted of methanol, and water was filtered before use by means of a 0.45μ membrane in a ratio of 60 to 40 composition of the mobile phase. The mobile phase was supplied with a flow rate of 1.0 ml/min from the solvent reservoir into the tank, and the injection volume was 20 μl. The temperature of the column was kept at 23 ± 10 °C. At 239 nm, eluent controls were performed. The optimized conditions were shown in Table 2.

Calibration of Standards: The standard calibration line was constructed for capecitabine. In order to achieve a concentration of 4-50 μg/ml of different capecitabine volumes of the stock, solutions have been correctly transferred to 10 ml volumetric cylindrical flasks. The reference line was achieved by diagraming the peak area of product concentration.

Method Validation: The method was validated according to the ICH guidelines. The following validations characteristics were addressed: specificity and selectivity, linearity, assay, accuracy, precision, limits of detection and quantitation, robustness, ruggedness ^10-12.

Specificity and Selectivity: Due to the presence of other excipients, the specificity of the method was assessed for interference. The figure shows that the medicinal product was clearly separated. This study therefore selects the HPLC method presented.

Linearity: The linearity of calibration curves in a pure solution (peak area vs.) was tested over the 4-50 μg/ml range. The regression lines for standard drug concentrations using an analysis of regression were lined with the calibration curves and y= 53752x+ 48825, r² = 0.991 for capecitabine. Total eluting time was less than 10 min. The mean ± standard difference (SD) was determined for the pitch, intercept, and correlation coefficient of the standard curves (N=3). Table 3-4 and Fig. 1 presented the data.

Precision: Six duplicate measurements of 20 μg/mL capecitabine concentration were tested for process accuracy; the results shown in Table 23. In order to determine process precision, intraday and inter-day measurements (days 3) of the drug were performed, using 8 μg/mL, 20 μg/mL, 40 mL, intra-day, and inter-day samples were calibrated with a standard curve simultaneously prepared during the day of testing by calibrating percent RSD for replicate samples (n=3). Table 7-8 revealed the intra-and inter-day accuracy tests.

Assay: The analysis was carried out by weighing 10 comprises a finely powdered tablet powder equivalent to the 10 mg of API and was precisely weighed. This was obtained with 10ml flask water and added water to label the solution with filtering paper from Whatman 1 (1000 mcg/ml). The volume was added with the moving phase (100mcg/ml), and 1ml was transferred into a 10 ml volumetric flask, then moved to the 10 ml volu-metric flask, and the volume upgraded to the mark with a moving phase (10 mcg/ml). The solution was sonicated for 15min, and the total area was measured under above chromatographic conditions.

The analysis process was done triple, and sample weight was determined for the test. Table 5 measured and displayed the percentage of products present in type, medium, and standard deviation.

Accuracy: Recovery tests have determined the reliability of the system. The drug's reference levels have been applied to the term at the rate of 50%, 100%, 150%. Recovery experiments have been conducted three times, and Table 6 and Fig. 2 have measured the percent recovery and relative standard deviation from the capecitabine recovery.

Limits of Detection and Quantitation: Samples containing very low analyte levels are carried out in compliance with the ICH Guidelines for the LOD and LOQ (Limit of Quantitation) detection for the procedure. Using the mathematical formulation process, LOD has been represented by setting the minimum level to reliably detect the analyte. LOQs in the criteria that can be calculated reproducibly with reasonable precision is known as the lowest concentration of analytes. Table 10 showed the LOD and LOQ values for Capecitabine.

Robustness: The method's robustness was assessed through examination of system suitability requirements and measurement of system suitability parameters by varying HPLC pump flow rates (±0.2%) and organic solvents (±2%) and mobile phase 60. No changes in the R.S.D. peak area, USP tailing factor and conceptual tiles caused any significant changes. While retention periods were much longer and statistical improvements were still possible. Table 9 revealed the results.

System Suitability: Tests are characterized as the methods that can produce an appropriate precision and accuracy. Following the development and validation of the process, the device suitability was performed. This was done by measuring parameters such as plate (N), resolution (R), tailor, capacity factor, HETP, sample maximum symmetry, and displayed in Table 11-12.

Stress Degradation Studies of Capecitabine:

Acid Degradation Studies: Acid decomposition studies were carried out in 0.1 N HCl at room temperature for a period of 3 days. 10mg of the standard capecitabine was weighed and transferred to separate 10 ml standard volumetric flasks. The drug was dissolved by adding water to get the final conc. of 1000μg/ml. From the above solutions, 1ml of capecitabine solution was drawn in to separate 10 ml std flasks and made up to the volume with 9 ml of 0.1N HCl to get the conc. of 100 μg/ml.

From this solution, 2 ml of capecitabine was drawn at time periods of 0 h, 1 h, 2 h, and 4 h, then added 4 ml of mobile phase and pH was adjusted to 4.0 with OPA. The volume was developed and injected with up to 10 ml mobile phase. As a controller, Blank was used (sample solution without the medicine) to determine mobile phase interference. Table 13-14 showed the findings.

Alkaline Degradation Studies: The analysis of alkaline decomposition at room temperature was carried out in 0.1 NaOH. The Capecitabine formulation weighed 10 mg and was moved to a generic 10ml volumetric container. The drugs are dissolved in water to achieve the final concentration of 1000 μg/ml 1 ml of capecitabine from the above solutions were drawn in regular 10ml containers, and the conc volume was made up of 0.1N NaOH 9 ml 100 μg/ml 100 μg. The 2 ml Cacpecitabine solution has been drawn at 0 h, 1 h, 2 h, and 4 h, and then 4 ml of the mobile stage has been applied, and the PH with OPA has been changed to 4.0. The amount was up to 10 ml and injected into the mobile phase. As a control, blank (sample solution without medicine) has been used, and mobile phase interference has been determined. Table 15-16 showed the results.

Neutral Condition: A separate 10-ml volumetric flask was drawn from the stock solution for standard capecitabine 1 ml, which is composed of the amount of 9 ml water for finally conc. 100 μg per ml. 1 ml of capecitabine was drawn and injected from these solutions into 10 ml volumetric flask, with a volume of 0 h, 1 h, 2 h, and 4 h.

Oxidative Degradation Studies: In order to achieve a final conc. of 10 ml volumetric flask, 1ml each was drawn from a stock solution with standard Capecitabine 100 microns/ml. 2 ml Capecitabine from these solutions were drawn in 10 ml volumetric bottle with the following dimensions: 0 h, 1 h, 2 h, and 4 h and made up to the volume with the mobile phase and injected. Stress specimens deteriorated from the highest areas in the chromatogram. The damaged substance content was determined by percentage. Table 17-18 shows the results. 0.3% H₂O₂ oxidative stress tests have been performed at room temperature.

Thermal Degradation Studies: Thermal degradation studies for both drug solutions were conducted by exposing the sample at 60 °C for 0 h, 1 h, 2 h and 4 h in hot air oven.

10 mg of capecitabine was measured into a different 10 ml bottle; the medicine was dissolved with a few ml of water and then diluted with air until the mark was over. Solution of 1000 μg/ml. 1 ml of capecitabine has been drawn from the above solutions into a 10 ml volumetric flask, up to a volume of water to achieve a final conc. 100 μg per liter. This solution was exposed to 60 °C and 2ml capecitabine was drawn to the final volume at 0 h, 1 h, 2 h and 4 h. Stress specimens suffered from the highest areas in the chromatogram, determined the percentage level of the damaged drug. Table 19-20 shows the findings. The results are shown.

Photolytic Degradation Studies: Photo stability degradation studies of capecitabine solution was carried out in a photostability chamber by exposing to UV light. Weighed 10 mg of capecitabine into 10 ml flasks and dissolved it with only a few ml liquid and diluted it with water to get eventually conc. of 1000 μg/ml. 1 ml of capecitabine has been drawn from the above solutions to 10 ml volumetric bottle and composed up to the volume of water to finish conc. 100μg/ml.

The solutions were drawn up and pumped into the last volume at 0 h, 1 h, 2 h and 4 h in the stability chamber with 2 ml capecitabine. Stress specimens were removed from the peak areas in the chromatogram. The deteriorated drug percentage was identified Table 21-22 show the results. The results are tabled.

RESULTS:

Validation of Analytical Method for the Assay of Capecitabine:

TABLE 2: OPTIMIZED CHROMATOGRAPHIC CONDITIONS OF CAPECITABINE

Specificity: Specificity is the ability of a method to discriminate between the intended analyte(s) and other components in the sample. Specificity of the HPLC method is demonstrated by the separation of the analytes from other potential components such as impurities, degradants, or excipients. Volume of 20 μL of working placebo sample solution was injected into the chromatograph and the chromatogram was recorded and presented below. No peaks were found at retention time of 7.927. As no peaks were found at retention time of 7.927, the proposed method was specific for the detection of capecitabine.

Linearity and Range:

TABLE 3: LINEARITY RANGE DATA FOR CAPECITABINE

FIG. 1: LINEARITY PLOT OF CAPECITABINE

TABLE 4: LINEARITY REPORT OF CAPECITABINE

Assay:

TABLE 5: ASSAY OF CAPECITABINE FORMULATION

Accuracy:

TABLE 6: RECOVERY REPORT OF CAPECITABINE

FIG. 2: RECOVERY PLOT OF CAPACETABINE

Precision:

TABLE 7: SYSTEM PRECISION DATA

TABLE 8: INTRA AND INTER-DAY PRECISION OF CAPECITABINE

Robustness:

TABLE 9: ROBUSTNESS STUDIES OF CAPECITABINE

Limit of Detection (LOD) and Limit of Quantification (LOQ)

TABLE 10: LOD AND LOQ REPORT OF DRUGS

System Suitability:

TABLE 11: SYSTEM SUITABILITY DATA OF CAPECITABINE

TABLE 12: SYSTEM SUITABILITY PARAMETERS FOR CAPECITABINE

Stress Degradation Studies for Capecitabine:

Degradation Behavior of Capecitabine:

Degradation in Acid:

TABLE 13: ACIDIC DEGRADATION OF CAPECITABINE AT ROOM TEMPERATURE

TABLE 14: DEGRADANTS FORMED DURING ACIDIC DEGRADATION AT ROOM TEMPERATURE

Discussion: The drug showed liability to acid hydrolysis at room temperature. It decomposed to the extent of 12.50% degradation in 0.1N HCl in 2 h. The degradation increased to 31.52% in 4 h.

Degradation in Base:

TABLE 15: ALKALI DEGRADATION OF CAPECITABINE AT ROOM TEMPERATURE

TABLE 16: DEGRADANTS FORMED DURING ALKALI DEGRADATION AT ROOM TEMPERATURE

Discussion: The drug showed liability to alkali hydrolysis at room temperature. It decomposed to the extent of 18.05% degradation in 0.1N NaOH in 2 h. The degradation increased to 32.92% in 4 h.

Oxidative Stress Study:

TABLE 17: OXIDATIVE DEGRADATION OF CAPECITABINE AT ROOM TEMPERATURE

TABLE 18: DEGRADANTS FORMED DURING OXI-DATIVE DEGRADATION AT ROOM TEMPERATURE

The drug showed highly liable to H₂O₂ at room temperature when compared to acid and alkali hydrolysis. It decomposed to the extent of 24.02% degradation in 0.3% H₂O₂ in 2 h. The degradation increased to 36.36% in 4 h.

Thermal Stress Study:

TABLE 19: THERMAL DEGRADATION OF CAPECITABINE SOLUTION AT NMT 60 °C

TABLE 20: DEGRADANTS FORMED DURING THER-MAL DEGRADATION OF SOLUTION AT NMT 60 °C

Discussion: The drug in solution form highly liable to thermal at temperature NMT 60 °C when compared to acid, alkali hydrolysis, and H₂O₂. The solution decomposed to the extent of 24.60% degradation in thermal in 2 h. The degradation increased to 42.41% in 4 h.

Photodegradation:

TABLE 21: PHOTODEGRADATION OF CAPECITABINE SOLUTION

TABLE 22: DEGRADANTS FORMED DURING PHOTO DEGRADATION OF CAPECITABINE SOLUTION

The drug showed higher liability to photodegradation at room temperature. It decomposed to the extent of 28.10% degradation in 2 h. The degradation increased to 57.24% in 4 h.

SUMMARY: In the present study, the API’s namely capecitabine (anticancer) the essential therapeutic agent in the treatment of cancer. Among the analytical techniques available in estimation and quantification HPLC method is an emerging technique reliable in vast areas of research that includes the other to undertake method development and validation as per ICH guidelines for the same.

In present work, forced degradation HPLC method has been developed for the estimation of capecitabine in marketed tablet formulation. Forced degradation The HPLC method was developed with the mobile phase system of MeOH: H₂O in the ratio of 60:40 v/v pH adjusted to 3 with orthophosphoric acid. The flow rate of 1ml/min was used on a C₁₈ column (250 × 4.6 mm, 5µm particle size). The retention time of capecitabine was observed at 6.97 min Table 23-24.

TABLE 23: SUMMARY OF VALIDATION PARAMETERS

TABLE 24: SUMMARY OF FORCED DEGRADATION STUDIES REPORT

The developed and validated stability indication HPLC method is found be linear, accurate, precise, specific, and robust. Hence the method can be used routinely for estimation of capecitabine in formulations.

CONCLUSION: The present work was aimed at developing new validated RP-HPLC method for selected API and application to stability-indicating assay. The work was aimed comparatively to the earlier literature report in connection to the priority of the present investigation with respect to the ICH guideline parameters. In the present study, the API´s namely capecitabine (anticancer) an essential therapeutic agent in the treatment of cancers.

Among the analytical techniques available in estimation and quantification HPLC method is an emerging technique reliable in vast areas of research that indicated the author to undertake method development and validation as per ICH guidelines for the same.

The present work developed the RP-HPLC method for capecitabine by standard external method using MeOH: H₂O (pH was adjusted to 3 with ortho-phosphoric acid) (60:40 v/v) as mobile phase and detection was performed at 239 nm with a retention time of 7.927 min and peak asymmetry of 1.04.

The method was validated for all validation parameters as per ICH guidelines. The linearity range for capecitabine was 4-50µg/ml, with r² value of 0.9991. The % RSD for intra and interday precision was < 2%. The method has been validated in the assay of tablet dosage forms. The accuracy of the method was validated by recovery studies and was found to significantly and under specification limits, with % recovery 99-101.5 (within the acceptable range 98-102%). The method also passed the specifications for robustness parameters.

A stability study on capecitabine was carried out, and an efficient HPLC method for quantification of capecitabine and identification of its degradation products in the bulk drug was developed and validated. The results of stress testing of the bulk drug, undertaken according to ICH guidelines, revealed that degradation products were formed under acidic, alkaline, thermal, oxidizing, and photolytic conditions.

ACKNOWLEDGEMENT: The writers are grateful to Dr. Y. Padmanabha Reddy, Principal, Dr. J. Ravindra Reddy, Vice-Principal and members of the Division of Pharmaceutical Analysis and Quality Assurance, Raghavendra Institute of Pharmaceutical Education and Research (RIPER), Anantapur for offering the required equipment, support and guidance during the thesis.

CONFLICTS OF INTEREST: Authors express no conflict of interest

REFERENCES:

1. Ramalakshmi S, Kavimani S, Srineevas S, Vetriselvi V and Bhaskar LVKS: Molecular Markers for Capecitabine Therapy: A Review. Int J Pharm Sci Res 2016; 7(11): 4315-26.
2. Anupama B, Jagathi V and Sai PP: Simultaneous estimation of gencitabine hydrochloride and capecitabine hydrochloride in combined tablet dosage form by RP-HPLC Method. E-Journal of Chemistry 2011; 8(3): 1212-17.
3. Sreevatsav ASK and Harishbabu AK: RP-HPLC method development and validation of Capecitabine Extended Release Tablet dosage form. Int J Pharm Sci Res 2013; 4(11): 4477-87.
4. Jayaseelan S, Bajivali SK, Ramesh U, Sekar V and Perumal P: Bioanalytical method Development and validation of Capecitabine by RP-HPLC method. International Journal of Chem Tech Research 2010; 2: 2086-90.
5. Hoff PM, Ansari R, Batist G, Cox J, Kocha W, Kuperminc M, Maroun J, Walde D, Weaver C, Harrison E and Burger HO: Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study. J Clin Oncol 2001; 19(8): 2282-92.
6. Narendra D, Saikishore Y, Pushpalatha P, Mamatha N and Venkatesh P: Development and validation of new RP HPLC method for analysis of Capecitabine in pharmaceutial dosage form. IJSIT 2013; 2(1): 21-30.
7. Rajitha K and Nagavalli D: Validated estimation of Capecitabine by UV-Spectroscopic, RP- HPLC and HPTLC method. IRJP 2012: 3(11): 163-66.
8. Panikumar AD, Raju YV, Sunitha G, Ramakrishna K, Ceema M and Venkateshwara RA: Development of validated stability indicating RP-HPLC method for the estimation of Capecitabine in pure and pharmaceutical formulations. International Journal of Research in Pharmaceutical and Biomedical Sciences 2011; 2(1): 175-81.
9. https://www.drugbank.ca/drugs/DB01101
10. Carr GP and Wahlich JC: A practical approach to method validation in pharmaceutical analysis. J. Pharm. Biomed. Anal 1990; 86: 613-18.
11. International Conference on Harmonization (ICH) Q2B. Validation of Analytical Procedures: Methodology; November 1996. Published in the Federal Register 1997; 62(96): 27463-67.
12. Monika B and Saranjit S: Development of validated stability-indicating assay methods-Critical review. Journal of Pharmaceutical and Biomedical Analysis 2002; 28: 1011-40.
    

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    How to cite this article:

    Lalitha KV, Reddy JR and Devanna N: Validated stability indicating HPLC method for estimation of capecitabine. Int J Pharm Sci & Res 2020; 11(11): 5651-58. doi: 10.13040/IJPSR.0975-8232.11(11).5651-58.

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