![IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750
w w w . i a j p s . c o m Page 551
INTRODUCTION:
Capecitabine is a fluoropyrimidine carbonate with
antineoplastic activity and it is in a class drugs known
as anti-metabolites. Capecitabine is an orally
administered chemotherapeutic agent used in the
treatment of metastatic breast and colorectal cancers
5'-deoxy-5-fluoro-N-[(pentyloxy) carbonyl] –
cytidine. Capecitabine is a prodrug of 5’-deoxy-5-
fluorouridine (5'-DFUR), which is enzymatically
converted to 5-fluorouracil in the tumour cells, where
it inhibits DNA synthesis and slows growth of
tumour tissue. The activation of Capecitabine follows
a pathway with three enzymatic steps and two
intermediary metabolites, 5'-deoxy-5- fluoro cytidine
(5'-DFCR) and 5'-deoxy-5-fluorouridine (5'-DFUR),
to form 5-fluorouracil. Capecitabine reached peak
blood levels in about 1.5hours (Tmax) with peak 5-FU
levels occuring slightly later, at 2hours. Food reduce
both the rate and extent of absorption of Capecitabine
with mean Cmax and AUC0-a decreased by 60% and
35%, respectively. The Cmax and AUC0-a of 5-FU
were also reduced by food by 43%and 21%
respectively. Food delayed Tmax of both parent and 5-
Fuby 1.5hours.Plasma protein binding of
Capecitabine and its metabolites is less than 60% and
is not concentration-dependent. Capecitabine was
primarily bound to human albumin (approximately
35%) Capecitabine is extensively metabolised
enzymaetically to 5-FU.the enzyme
dihydropyrimidine dehydrogenase hydrogenates 5-
FU, the product of Capecitabine metabolism, to the
much less toxic 5-fluoro 5, 6 dihydro-fluorouracil
(FUH2) dihydropyrimidinase claves the pyrimidine
ring to yield 5-fluorto-ureido propionicacid (FUPA).
Finally, b-ureido-pripionase cleaves FUPA to a-
fluoro-b-alanine (FBAL) which is cleared in the
urine.
MATERIALS AND METHODS:
Chemicals and Reagents: HPLC grade acetonitrile,
methanol.0.1N NaOH and water were purchased
from Hetero Labs Ltd, Jadcherla, and Hyderabad,
India.Preparation of standard solutions: About 100
mg of Capecitabine was accurately weighed and
transferred into a 100 ml volumetric flask and diluted
to volume with methanol to get the stock solution.
This gave a concentration of 1000 μg / ml.
Preparation of working stock solutions: 0.6 ml of
stock solution was pipetted out and placed in a 10 ml
volumetric flask and the volume was made up to
mark with methanol. This gave a solution containing
60μg/ml.
Preparation of mobile phase: A mixture of
Methanol, acetonitrile and water (80:18:2 v/v) was
employed as a mobile phase. 400 ml of methanol, 90
ml of ACN and 10 ml of water was mixed and
sonicated for 15 min. This was filtered by using a
0.45 μm filter paper.
Sample preparation: Ten tablets were weighed and
finely powdered. The powder equivalent to 100 mg
of Capecitabine accurately weighed and transferred
to volumetric flask of 100 ml capacity containing 25
ml of the methanol and sonicated for 15 min. The
flask was shaken and volume was made up to the
mark with methanol to give a solution of 1000 µg/ml.
The above solution was filtered through 0.45 µm
filter paper. From this solution 0.6 ml was diluted to
10 ml with methanol to give a solution 60 µg/ml.
Determination of λmax by UV spectrophotometer:
The standard solutions of Capecitabine were scanned
in the range of 200-400nm against methanol as a
blank. Capecitabine showed maximum absorbance at
240 nm. So the wavelength selected for the
determination of Capecitabine was 240 nm.
RESULTS AND DISCUSSION:
Method development: HPLC chromatographic
separation were carried out in an isocratic mode by
using the following optimized conditions and the
corresponding chromatogram.
Chromatographic conditions:
Mobile phase: Filter and degassed mixer of water
and acetonitrile in the ratio of 80 : 20
Column: Nova pack 300x3.9 mm 5 m
Flow rate: 1.0 ml /minute
Wavelength: 240 nm
Load: 10 µl
Run time: 60 min
Column temperature: 35 °C
Sample preparation: Weigh 25 mg of Capecitabine
WS in 50ml volumetric flask dissolve and dilute to
50ml with mobile phase. The retention time
Capecitabine is about 20.0 min. In this trial all
impurities are separated from the main peak. The
specificity test of the proposed method was
demonstrated by blank interference i.e. the blank
chromatogram did not interfered with that of the drug
peak.](https://image.slidesharecdn.com/9-170424055911/85/DEVELOPMENT-AND-VALIDATION-OF-CAPECITABINE-TABLET-PHARMACEUTICAL-DOSAGE-FORM-BY-USING-RP-HPLC-METHOD-2-320.jpg)
DEVELOPMENT AND VALIDATION OF CAPECITABINE TABLET (PHARMACEUTICAL DOSAGE FORM) BY USING RP-HPLC METHOD.
- 1. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 550 CODEN (USA): IAJPBB ISSN: 2349-7750 IINNDDOO AAMMEERRIICCAANN JJOOUURRNNAALL OOFF PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS http://doi.org/10.5281/zenodo.400638 Available online at: http://www.iajps.com Research Article DEVELOPMENT AND VALIDATION OF CAPECITABINE TABLET (PHARMACEUTICAL DOSAGE FORM) BY USING RP-HPLC METHOD. Ananda Kumar. Chettupalli*, Vivek Kunduru, Narender Boggula, Vasudha Bakshi Department of Pharmaceutics, Anurag Group of Institutions, Venkatapur, Gatkesar, Medchal, Telangana, India- 500088. Received: 27 February 2016 Accepted: 09 March 2017 Published: 14 March 2017 Abstract: A new precise accurate and reliable validated method for the determination of Capecitabine was developed by using reverse phase high performance liquid chromatography in pharmaceutical dosage forms. Spectrophotometer determination was carried out at an absorption maximum of 240nm by using methanol. The linearity was over the concentration range of 20-120 µg/ml with correlation coefficient 0.999. Chromatographic separation was carried out by using a mobile phase of methanol: Acetonitrile: water (80:20:80 V/V) on Waters 2487 dual absorbance column in an isocratic mode at a flow rate of 1.1 ml/min with UV detection at 240 nm. The developed methods were found to be precise and accurate for the estimation of Capecitabine in pharmaceutical dosage forms and could be used for routine analysis. Keywords: Capecitabine, RP-HPLC, Spectrophotometry, Waters 2487 dual absorbance detector, Nova pack 300 × 3.9mm 5μ as column, 240nm Corresponding Author: Ananda Kumar. Chettupalli Department of pharmaceutics, Anurag Group of Institutions, Venkatapur, Gatkesar, Medchal, Telangana, India- 500088. Email: anand33.chettupalli@gmail.com Ph No: 9849922424 Please cite this article in press as Ananda Kumar. Chettupalli et al, Development and Validation of Capecitabine Tablet (Pharmaceutical Dosage Form) By Using RP-HPLC Method, Indo Am. J. P. Sci, 2017; 4(03). QR code
- 2. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 551 INTRODUCTION: Capecitabine is a fluoropyrimidine carbonate with antineoplastic activity and it is in a class drugs known as anti-metabolites. Capecitabine is an orally administered chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers 5'-deoxy-5-fluoro-N-[(pentyloxy) carbonyl] – cytidine. Capecitabine is a prodrug of 5’-deoxy-5- fluorouridine (5'-DFUR), which is enzymatically converted to 5-fluorouracil in the tumour cells, where it inhibits DNA synthesis and slows growth of tumour tissue. The activation of Capecitabine follows a pathway with three enzymatic steps and two intermediary metabolites, 5'-deoxy-5- fluoro cytidine (5'-DFCR) and 5'-deoxy-5-fluorouridine (5'-DFUR), to form 5-fluorouracil. Capecitabine reached peak blood levels in about 1.5hours (Tmax) with peak 5-FU levels occuring slightly later, at 2hours. Food reduce both the rate and extent of absorption of Capecitabine with mean Cmax and AUC0-a decreased by 60% and 35%, respectively. The Cmax and AUC0-a of 5-FU were also reduced by food by 43%and 21% respectively. Food delayed Tmax of both parent and 5- Fuby 1.5hours.Plasma protein binding of Capecitabine and its metabolites is less than 60% and is not concentration-dependent. Capecitabine was primarily bound to human albumin (approximately 35%) Capecitabine is extensively metabolised enzymaetically to 5-FU.the enzyme dihydropyrimidine dehydrogenase hydrogenates 5- FU, the product of Capecitabine metabolism, to the much less toxic 5-fluoro 5, 6 dihydro-fluorouracil (FUH2) dihydropyrimidinase claves the pyrimidine ring to yield 5-fluorto-ureido propionicacid (FUPA). Finally, b-ureido-pripionase cleaves FUPA to a- fluoro-b-alanine (FBAL) which is cleared in the urine. MATERIALS AND METHODS: Chemicals and Reagents: HPLC grade acetonitrile, methanol.0.1N NaOH and water were purchased from Hetero Labs Ltd, Jadcherla, and Hyderabad, India.Preparation of standard solutions: About 100 mg of Capecitabine was accurately weighed and transferred into a 100 ml volumetric flask and diluted to volume with methanol to get the stock solution. This gave a concentration of 1000 μg / ml. Preparation of working stock solutions: 0.6 ml of stock solution was pipetted out and placed in a 10 ml volumetric flask and the volume was made up to mark with methanol. This gave a solution containing 60μg/ml. Preparation of mobile phase: A mixture of Methanol, acetonitrile and water (80:18:2 v/v) was employed as a mobile phase. 400 ml of methanol, 90 ml of ACN and 10 ml of water was mixed and sonicated for 15 min. This was filtered by using a 0.45 μm filter paper. Sample preparation: Ten tablets were weighed and finely powdered. The powder equivalent to 100 mg of Capecitabine accurately weighed and transferred to volumetric flask of 100 ml capacity containing 25 ml of the methanol and sonicated for 15 min. The flask was shaken and volume was made up to the mark with methanol to give a solution of 1000 µg/ml. The above solution was filtered through 0.45 µm filter paper. From this solution 0.6 ml was diluted to 10 ml with methanol to give a solution 60 µg/ml. Determination of λmax by UV spectrophotometer: The standard solutions of Capecitabine were scanned in the range of 200-400nm against methanol as a blank. Capecitabine showed maximum absorbance at 240 nm. So the wavelength selected for the determination of Capecitabine was 240 nm. RESULTS AND DISCUSSION: Method development: HPLC chromatographic separation were carried out in an isocratic mode by using the following optimized conditions and the corresponding chromatogram. Chromatographic conditions: Mobile phase: Filter and degassed mixer of water and acetonitrile in the ratio of 80 : 20 Column: Nova pack 300x3.9 mm 5 m Flow rate: 1.0 ml /minute Wavelength: 240 nm Load: 10 µl Run time: 60 min Column temperature: 35 °C Sample preparation: Weigh 25 mg of Capecitabine WS in 50ml volumetric flask dissolve and dilute to 50ml with mobile phase. The retention time Capecitabine is about 20.0 min. In this trial all impurities are separated from the main peak. The specificity test of the proposed method was demonstrated by blank interference i.e. the blank chromatogram did not interfered with that of the drug peak.
- 3. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 552 Fig 1:Purity plot of Acid stressed Capecitabine tablets Fig 2: Purity plot of Thermal heat stressed Capecitabine tablets Linearity and range: Chromatographic method was tested for linearity by plotting peak area against concentration of solution. Linearity ranges and correlation coefficients obtained were presented in Table 1. TABLE 1: LINEARITY OF TEST METHOD Spike level Average Amount added (in mg/ml) Average Amount recovered (in mg/ml) 25% 0.0260 0.0265 50% 0.0502 0.0509 100% 0.1014 0.1034 150% 0.1544 0.1588 200% 0.1953 0.1965
- 4. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 553 Linearity of Test Method 0 0.05 0.1 0.15 0.2 0.25 0.000 0.050 0.100 0.150 0.200 0.250 Response Conc Method Linearity Fig 3: Linearity of Test Method Accuracy of the method: To ensure the reliability and accuracy of the method, the recovery studies were carried out by adding a known quantity of drug with pre-analyzed sample and contents were reanalyzed by the proposed method. Accuracy was evaluated by injecting triplicate injections at three different concentrations levels equivalent to 25%,50 %, 100 %,150 % and 200% of the active ingredient, by adding a known amount of Capecitabine standard to a sample of known concentration and calculating the recovery of Capecitabine with % RSD and % recovery for each concentration. The mean %recoveries were in between 98.22-101.49% and were given in table2. TABLE 2: Accuracy of Capecitabine Sample No. Spike level ‘mg/ml’ added ‘mg/ml’ found Mean % Recovery 1. 25% 0.0258 0.0265 102.8 101.72. 25% 0.0257 0.0262 101.9 3. 25% 0.0266 0.0267 100.4 4. 50% 0.0504 0.0514 101.9 101.35. 50% 0.0495 0.0500 101.0 6. 50% 0.0507 0.0512 100.9 7. 100% 0.1025 0.1053 102.7 101.98. 100% 0.1011 0.1027 101.6 9. 100% 0.1007 0.1022 101.5 10. 150% 0.1519 0.1564 102.9 102.811. 150% 0.1549 0.1592 102.8 12. 150% 0.1568 0.1608 10.28 13. 200% 0.1950 0.1963 100.6 100.614. 200% 0.1975 0.1998 101.2 15. 200% 0.1933 0.1934 100.1
- 5. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 554 The Mean recovery of Capecitabine at each spike level should be not less than 97.0% and not more than 103.0%. The Mean recovery of Capecitabine at each spike level is within the specification. Precision of the method: The intra-day and inter-day variations of the method were determined by using six replicate injections of one concentration and analysed on the same day and three different days over a period of two weeks. The result revealed that, the precision with %RSD (0.66% and 1.02%) respectively for intra-day and inter-day. Results of intra-day and inter-day precision studies are shown in table3. The relative standard deviation for six % assay results should be not more than 2.0% for both the Analysts. The Assay of Capecitabine should be not less than 97.0% and not more than 103.0%. The observed assay results of Capecitabine are within in the specifications. Limit of detection (LOD) and Limit of quantification: To determine the Limit of Detection the sample was dissolved by using Mobile Phase and injected until peak was disappeared. After 2µg/ml dilution, Peak was not clearly observed. So it confirms that 0.625 µg/ml is limit of Detection. And Limit of Quantification found to be 2µg/ml. For this study six replicates of the sample at lowest concentration were Measured and quantified. The LOD and LOQ were found to be 0.6µg/ml and 2µg/ml respectively. TABLE 3: Precision of Capecitabine System suitability Parameters Observed value Acceptance Criteria Analyst – 1 Analyst- 2 Tailing factor of Capecitabine 1.52 1.51 NMT 2.0 Relative standard deviation of Capecitabine area from five replicate injections of standard 0.49 0.11 NMT 2.0% INTERMEDIATE PRECISION (ANALYST TO ANALYST VARIATION) TABLE 4: INTERMEDIATE PRECISION Sample No. Assay of Capecitabine as % of labeled amount Analyst – 1 Analyst – 2 1 99.0 99.3 2 98.3 98.7 3 99.2 98.5 4 98.5 99.1 5 98.8 98.4 6 98.3 98.1 Mean 98.68 98.7 % RSD 0.4 0.5
- 6. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 555 Robustness: The robustness study was performed by slight modification in flow rate and composition of the mobile phase. Capecitabine at 60µg/ml concentration was analyzed under these changed experimental conditions. In this study, the chromatographic parameters monitored were, retention time, area, capacity factor, tailing factor and theoretical plates. The obtained results of robustness study are shown in table5. The difference of % assay result from centrifuged sample to filtered samples should be not more than 3.0 the differences of % assay results from centrifuged sample to filtered samples are within the specification Ruggedness: Inter day variations were performed by using six replicate injections of standard solution of concentrations which were prepared and analyzed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of percentage relative standard deviation. TABLE 5: System Suitability Parameters (robustness study) System Suitability Parameters Observed value Acceptance Criteria 90% of method organic phase 100% of method Organic phase 110% of method Organic phase Tailing factor 1.48 1.52 1.54 NMT 2.0 RSD of five injections of standard 0.06 0.10 0.06 NMT 2.0% System Suitability Parameters Observed value with Flow rate Acceptance Criteria 0.8 mL/min 1.0 mL/min 1.2 mL/min Tailing factor 1.61 1.51 1.45 NMT 2.0 RSD five injections of standard 0.52 1.20 0.26 NMT 2.0%
- 7. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 556 Centrifuged Sample filtered through PVDF filter Sample filtered through Nylon 66 filter % Assay % Assay Difference % Assay Difference 98.8 98.9 100.7 98.5 1.9 0.4 98.5 96.9 0.3 2.0 TABLE 6: System Suitability Parameters (Ruggedness) System suitability Parameters Observed value Acceptance Criteria System – 1 System – 2 Tailing factor of Capecitabine peak 1.52 1.36 NMT 2.0 Relative standard deviation of Capecitabine peak area from five replicate injections of standard 0.49 0.3 NMT 2.0% TABLE 7: Assay of Capecitabine Sample No. Assay of Capecitabine as % of labeled amount System – 1 System – 2 1 99.0 98.7 2 98.3 98.8 3 99.2 98.0 4 98.5 98.4 5 98.8 98.6 6 98.3 99.0 Mean 98.68 98.58 RSD 0.4 0.4 System suitability parameters: The system suitability tests were carried out on freshly prepared standard solution (60µg/ml) of Capecitabine under the optimized chromatographic conditions .from that the parameters that were studied to evaluate the suitability of the system were: a) No. of theoretical plates b) tailing factor c) retention time. CONCLUSION: In this thesis the author made a humble attempt in developing “New RP-HPLC methods for estimation of assay of Capecitabine tablet and its known impurities” with the facilities, and the results are incorporated in this thesis. Opens with the general introduction, aim and plan of work introduction to drug and its known impurities used in the present study, Introduction to HPLC, General guidelines for HPLC method development and guidelines for method validation. The results indicate that the proposed methods are sensitive, accurate, precise, simple and reproducible and can be used for routine determination of Capecitabine tablets and its impurities in bulk drug samples and pharmaceutical formulations.
- 8. IAJPS 2017, 4 (03), 550-557 Ananda Kumar. Chettupalli et al ISSN 2349-7750 w w w . i a j p s . c o m Page 557 REFERENCES: 1.www.drugbank.com 2.Indian Pharmacopoeia. Vole 2. 2010. p. 972-3 3.Lange. Basic and clinical pharmacology. 11thed:p.947. 4.A.H. Beckett, J.B. Stenlak, Practical Pharmaceutical Chemistry, C.B.S.Publications. 5.Douglas A.Skoog., Donal M.West., Fundamentals of Analytical Chemistry, 7th edition. 6.Hohat H.Willard., Lynne L. Merrit, John A. Dean., Instrumental methods of analysis, 7th edition, CBS Publishers, New Delhi. 7.Lloyd r. Snyder, Joseph J.Kirkland, Joseph L.Glajeh, Practical HPLC method development, 2nd edition,1-14,1997. 8.L.R.Snyder, P.W.Carr and S.C. Rutan, J.Chromatogr.A, 656, 537 (1993) 9.R.J.Hamilton and Swell, Introduction to HPLC, 2nd edition, 2-94. 10.Sharma, B.K., Instrumental methods of Chemical analysis, 19th edition, 2000.