Int J App Pharm, Vol 8, Issue 3, 2016, 24-29Original Article


DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD AND RP-HPLC METHOD FOR ESTIMATION OF CAPECITABINE IN BULK AND TABLET DOSAGE FORMS

K. PALLAVI *, P. SRINIVASA BABU, G. KISHORE BABU

Department of Pharmaceutics, Vignan Pharmacy College, Vadlamudi, Guntur (dt.), ap, 522213
Email: pallavi1203@gmail.com         

Received: 21 Mar 2016, Revised and Accepted: 19 Jun 2016


ABSTRACT

Objective: The objective of the present work is to develop and validate a novel, specific, precise and reliable method for estimation of Capecitabine in bulk and pharmaceutical dosage form using UV-visible spectroscopy and RP-HPLC method.

Methods: UV-visible spectrophotometric determination was performed with Elico double beam SL 210 UV-visible spectrophotometer having deuterium lamp at λmax 304 nm using water as a medium. Linearity was noted over a concentration range of 2-20 µg/ml with a correlation coefficient of 0.99. HPLC analysis was performed using Eclipse XDB C18 column with 5 µm particle size having dimensions 4.6 X 250 mm column, Agilent 1260 infinity DAD detector, 1260 infinity quaternary pump using Ezchrome software at a flow rate of 1 ml/min and a run time pressure of 2140 psi. Methanol: acetonitrile: water in the ratio 30: 30: 40 was used as mobile phase and the effluents were analyzed at 304 nm. Both the proposed methods were validated for various parameters like linearity, precision, accuracy, robustness, ruggedness, selectivity, detection, quantification limits, formulation analysis as per International Conference on Harmonization (ICH) guidelines.

Results: Linearity for UV and HPLC method was noted over a concentration range of 2-100 µg/ml with a correlation coefficient of 0.99. The retention time was considered to be 4.60 min. The % RSD for interday and intraday precision studies and recovery analysis of both UV and HPLC methods was found to be less than 1% which is less than the official RSD limit (2%). Recovery analysis performed using marketed formulation capeguard was considered to be greater than 99% for both the methods.

Conclusion: Both the methods developed were validated according to the ICH guidelines. Hence it was evident that the developed methods were novel, sensitive, precise and reliable for estimation of Capecitabine in bulk and were successfully applied for estimation of pharmaceutical dosage forms.

Keywords: Capecitabine, UV-visible spectroscopic method, HPLC method, Validation


INTRODUCTION

Capecitabine (CPTB) is a new, orally administered, enzyme-activated fluoropyrimidine carbamate belonging to the class antimetabolites. The drug is official in the Indian Pharmacopoeia (IP)[1, 2]. The chemical name of Capecitabine is 5-deoxy-5-fluoro-N-(pentyloxy) carbonyl]–cytidine with a molecular formula C15H22FN3O6 and molecular weight 359.35[3, 4]. The chemical structure is intended to generate high levels of fluorouracil (5-FU) in tumor cells located in the colorectal and breast region. Capecitabine is a prodrug of 5-deoxy-5-fluorouridine (5-DFU), which undergoes enzymatic conversion to be 5-fluorouracil [5-FU] in tumor cells [5, 6]. The 5-FU inhibits the synthesis of thymidine monophosphate, which is an active form of thymidine required for de novo synthesis of Deoxy Ribo Nucleic acid [7-10]. The empirical formula of the functional moiety is C15H22FN3O6. The active moiety acts by mimicking as a normal cell nutrient which is indispensable to the growth of cancer cells. Literature survey disclosed that there are few analytical methods for spectrophotometric [11] and chromatographic estimation of capecitabine [12-16].

A detailed review of the literature regarding the existing methods revealed that there is a need for the development of the spectrophotometric method and chromatographic method, which is simple with less time tedious mobile phase or dilution medium. In the present study modest, novel, sensitive, reliable and accurate methods have been established for the estimation of capecitabine in bulk and in tablet dosage forms. Both the methods have a simple medium like water or feasible mobile phase mixture, which can be used for analysis of capecitabine with better separation of analytes. An attempt was made in the present methods to achieve an accurate, reliable and reproducible result with minimum Relative Standard Deviation (RSD) values than all other existing methods, which was successfully accomplished.

MATERIALS AND METHODS

Chemicals and reagents

The reference sample capecitabine (CPTB) was secured from Torrent Pharmaceuticals Limited. Ahmedabad. All the reagents used stood at analytical grade. Acetonitrile (HPLC grade) and triethylamine (HPLC grade) were acquired from Merck pharmaceutical's private ltd., Mumbai, India. Methanol and water used were of HPLC grade and purchased from Merck specialty's private ltd., Mumbai, India. Commercial tablet CAPEGUARD was procured from the local market.

Instrument specifications

The UV analysis was performed using Elico double beam SL 210 UV-visible spectrophotometer having deuterium lamp associated with spectra treats software.  The HPLC analysis was performed using agilent 1260 infinity system (Ezchrome elite software) consisting of DAD VL detector adjusted to a wavelength of 304 nm. The instrument also consisted of an eclipse XDB model C18 column (5 µm particle size, 4.6 x 250 mm) and a 1260 infinity VL quaternary pump.

Spectrophotometric and chromatographic conditions

Spectrophotometric analysis was performed using triple distilled water as mobile phase. The detection was carried out at an absorption maximum (λmax) of 304 nm.

Chromatographic separation was achieved using mobile phase methanol: acetonitrile: water at a ratio 30: 30: 40. A flow rate of 1 ml/min was maintained throughout the separation process. Run time pressure of 2140 psi was maintained. All the contents of the mobile phase were filtered through a 0.45 mm membrane filter and degassing was performed using ROHS sonicator to remove dissolved gases if any. For each trial, 20 µl samples were injected manually, and a total run time of 6 min was maintained. The eluent was detected at 304 nm. Various systems suitability parameters were assessed as mentioned in table 1.

Table 1: System suitability parameters for HPLC

S. No.

Parameter

Results

1

Standard concentration

1 mg/ml

2

Mobile phase

30:30:40 Methanol: acetonitrile: water

3

Elution

Isocratic

4

Wavelength

304 nm

5

Column

Eclipse XDB C18

6

Detector

DAD PDI detector

7

Flow rate

1 ml/min

8

Column Volume

1322.5 mm3

9

Run time pressure

2140 psi

10

Retention time

4.6 min

11

Runtime

6 min

12

Peak area

4438704.660

13

Tailing factor

0.941

14

Peak Asymmetry Factor

0.882

15

Column dead time

1322.5 min

16

Purging valve pressure

140 psi


Preparation of stock solutions and sample solutions

For the spectrophotometric analysis stock solutions of CPTB was prepared by dissolving 10 mg of the drug in 10 ml distilled water to obtain a final concentration of 1 mg/ml. Serial dilutions were made to prepare diverse sample solutions of concentrations ranging from 2–20 µl/ml. The solutions were analyzed at an absorption maximum of 304 nm against the blank.

For chromatographic analysis of CPTB, the stock solution was prepared by dissolving 10 mg of the drug in 10 ml of the mobile phase to obtain a final concentration of 1 mg/ml. Serial dilutions were made to obtain sample solutions of concentrations ranging from 2–100 µl/ml. All the sample solutions were filtered through 0.45 mm membrane filters and were subjected to degassing. The sample solutions were analyzed at an absorption maximum of 304 nm.

Validation of developed methods [17-19]

Linearity and range

Linearity is defined as the ability to obtain test results, which were directly proportional with the concentration of an analyte in the sample within a given range.

Linearity data for the spectrophotometric method was obtained at an absorption maxima of 304 nm as shown in fig. 1 by using ten concentrations in the range of 2–20 µg/ml. Calibration curve was obtained by plotting absorbance against concentration by considering six observations as shown in fig. 2.

Linearity data for the chromatographic method was obtained by using ten concentrations within the range of 2–100 µg/ml. Calibration curve was obtained plotting peak area against concentration by considering five observations as shown in fig. 3.

Both the methods were studied using six replicates of each sample concentrations.

Precision

The degree of closeness of agreement between a series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed condition was determined. The intra-day precision was performed by analyzing six replicate standard solutions on the same day, and inter-day precision was performed by analyzing a series of standard solutions for 3 consecutive days using the proposed UV and HPLC methods. The data obtained was presented in table 5.

Robustness

Robustness is defined as the measure of its capacity to remain unaffected by small but deliberate variation in method parameters, and it provides an indication to its reliability during normal range. Robustness of both the methods was studied using six replicates of the sample at a concentration level of 60 µg/ml (for HPLC) and 10 µg/ml (for UV).

Ruggedness

Ruggedness was calculated by considering the same sample at different labs by different analysts.

Detection and quantification limits

Limit of detection (LOD) represents the lowest amount of analyte in the sample which can be detected.

Limit of quantification (LOQ) represents the lowest amount of analyte, which can be quantitatively determined.

The above parameters are calculated based on the standard deviation of the response and the slope. The standard deviation was calculated based upon the calibration curve.

LOD = 3.3σ/SLOQ = 10σ/S

Selectivity and specificity

The ability to measure accurately and specifically the analyte of interest in the presence of other components like excipients in the tablet formulation was analyzed. The blank, standard, placebo, placebo along with analyte and test preparations were analyzed as per the method to identify interference of blank and placebo with CPTB peaks.

Estimation of an active ingredient in bulk and in tablet dosage form (Formulation analysis)

Twenty tablets (capeguard 500 mg) were weighed accurately and crushed into powder form. Accurately weighed the quantity of powder taken and a standard solution of 1000 µg/ml was prepared using the mobile phase and the diluting fluid. Serial dilutions were taken to ensure the standard solution prepared, and the solutions were analyzed spectrophotometrically and chromatographically using the proposed methods.

RESULTS AND DISCUSSION

Linearity and range

The linearity of CPTB employing UV method was constructed by considering concentration (µg/ml) on X–axis and Absorbance on Y–axis. The regression coefficient was considered to be 0.999 over a concentration range of 2–20 µg/ml. The representative linearity equation was found to be y = 0.0256x+0.0002 as showed in fig. 2 and data were shown in table 3.

The linearity of proposed CPTB employing HPLC method was constructed by considering concentration (µg/ml) on X–axis and peak area on Y-axis. The regression coefficient was considered to be 0.999 over a concentration range of 2–100 µg/ml. The representative linearity equation was found to be y = 57534x+17091 as showed in fig. 3 and the corresponding data were shown in table 3.

For both the methods the % RSD was found to be within the acceptable theoretical limits of ≤ 2%.

Table 3: Linearity data table for proposed HPLC and UV methods (where n=6)

HPLC linearity data

UV linearity data

Concentration (mcg/ml)

Peak area±RSD

Concentration (mcg/ml)

Absorbance

2

158716±639.22

2

0.049±0.005

4

262784±12119.81

4

0.106±0.011

6

425330±2064.75

6

0.158±0.006

8

528899±66248.83

8

0.207±0.002

10

638175±1820.09

10

0.258±0.006

20

1167819±60367.12

12

0.303±0.002

40

2266267±100287.5

14

0.355±0.006

60

3468807±374512.7

16

0.415±0.008

80

4496095±3019.35

18

0.463±0.013

100

5885228±139667

20

0.514±0.011

Correlation coefficient

0.999

Correlation coefficient

0.999

Slope

57300

Slope

0.025

Intercept

35375

Intercept

0.000


Fig. 1: UV visible spectrum scan of CPTB in water and methanol


Fig. 2: Linearity curve of CPTB obtained from UV method


Fig. 3: Linearity curve of CPTB obtained from HPLC method

Precision

The % RSD for intra-day precision (six independent series in the same day) and inter-day precision (3 consecutive days) analysis performed for six different individual samples of drug solution using the proposed UV and HPLC methods was found to be 0.021%, 0.72% and 0.39%, 0.91% respectively. Since the values obtained as shown in table 4 were within the proposed theoretical limits<2% RSD according to IP, the method was demonstrated to be precise.

Table 4: Precision analysis data of CPTB for UV and HPLC

Parameter

UV

HPLC

Interday (% RSD)

0.021

0.39

Intraday (% RSD)

0.72

0.91


Recovery studies

The accuracy of the proposed UV-visible spectroscopic method and HPLC method was established by recovery experiments. The recovery analysis studies were carried out at three different concentration ranges (50, 100 and 150%). All studies were carried in triplicate, and the results obtained were presented in table 5. The analyzed samples yielded high recovery values from the proposed methods. % RSD values were found to be less than 0.2% for both UV and HPLC analysis, indicating that the proposed methods were accurate. All the RSD values obtained were less than the theoretical limit of<2% RSD according to IP. F-test results for both the UV and HPLC methods revealed that the Fcal value is less than the Ftabulated value as shown in table 6 & 7, proving that null hypothesis is accepted. Hence it was proved that there is no significant difference between the actual amount added, and the amount recovered.

Table 5: Recovery analysis for CPTB by the proposed UV and HPLC methods

Method

Concentration level

Amount added (µg/ml)

Total amount (µg/ml)

Amount found (µg/ml)

Amount recovered (µg/ml)

% recovery

% RSD

UV

50

5

15

14.97

4.97

99.4

0.054

100

10

20

19.73

9.81

98.1

0.063

150

15

25

24.82

14.78

98.53

0.059

HPLC

50

5

15

14.94

4.94

98.8

0.108

100

10

20

19.73

9.73

97.3

0.038

150

15

25

24.82

14.82

98.8

0.092


 

Table 6: Single factor ANOVA for recovery studies performed using UV method

Source of variation

SS

df

MS

F cal

P-value

F tab

Between Groups

0.032266667

1

0.032267

0.001315

0.972806

7.708647

Within Groups

98.12086667

4

24.53022


Table 7: Single factor ANOVA for recovery studies performed using HPLC method

Source of variation

SS

Df

MS

F cal

P-value

F tab

Between Groups

0.04335

1

0.04335

0.001755

0.968595

7.708647

Within Groups

98.8222

4

24.70555


Robustness

The robustness of the proposed HPLC method was checked in terms of variation in mobile phase, flow rate change and wavelength change. Experimental findings proved that the change of mobile phase is the most influential factor on repeatability of the proposed HPLC method. Suitable measures have been adopted to maintain similarity in various instrumental aspects like injection and capillary conditioning. Since % RSD values for all the parameters were found to be less than 0.1% (less than the acceptable theoretical limit of<2% RSD) the proposed HPLC method was found to be robust. The results obtained were presented in table 8.

The robustness of the proposed UV method was checked in terms of variation in mobile phase and change in wavelength. Experimental findings proved that change in the mobile phase has a higher influence on repeatability of the proposed UV method compared to change in wavelength. % RSD values for all the parameters were found to be less than 0.02% (less than the acceptable theoretical limit of<2% RSD) which proved that the proposed UV method was found to be robust. The results obtained were presented in table 9.

Table 8: Results obtained for robustness study of HPLC method (n=6)

S. No.

Parameter

Condition

Area±RSD

% of change

1

Standard solution (60 mcg)

Standard condition

3468807

----

2

Mobile phase change

Methanol: acetonitrile: water 28: 32: 80

3223986±1023.47

0.071

Methanol: acetonitrile: water 30: 28: 78

3733627±1945.68

0.076

3

Flow change

0.9 ml/min

3592787±2012.63

0.036

1.1 ml/min

3686436±1897.13

0.063

4

Wavelength change

302 nm

3492661±1895.85

0.007

306 nm

3447989±2147.58

0.006


Table 9: Results obtained for robustness study of UV-visible spectrophotometric method (n = 6)

S. No.

Parameter

Condition

Absorbance (nm)

% of change

1

Standard solution (10 mcg)

Standard condition

0.258±0.017

----

2

Mobile phase change

Distilled water: methanol (98: 2)

0.261±0.047

0.0114

Distilled water: methanol (97: 3)

0.262±0.053

0.0153

3

Wavelength change

302 nm

0.256±0.068

0.007

306 nm

0.259±0.038

0.004

Table 2: Summary of validation parameters obtained for proposed UV and HPLC methods

Validation parameters

UV

HPLC

Beer's law limit

2-20

2-100

Correlation coefficient (r2)

0.999

0.999

Regression equation

Y = 0.025x+0.000

y = 57283x+37336

Slope

0.025

57283

Intercept

0.000

37336

LOD

0.234

0.097

LOQ

0.710

0.294


 

Ruggedness

Standard solutions of CPTB were analyzed using both the proposed methods for ruggedness.

The results showed that there is not any significant statistical difference between labs, analysts or between instruments. Thus both the methods are proven to have ruggedness.

Detection and quantification limits

The LOD and LOQ for CPTB utilizing the proposed UV method were determined to be 0.234 µg/ml and 0.710 µg/ml respectively.

The LOD and LOQ for CPTB using the proposed HPLC method were found to be 0.0969µg/ml and 0.2936µg/ml respectively. The results obtained were presented in table 2.

Both the methods indicate the accuracy and precision to detect a very low quantity of analyte which is a favorable sign for extending the method to plasma drug analysis.

Specificity

The selectivity and specificity of the proposed methods were tested by studying the effect of various excipients and other additives usually present in the formulations of cCPTB. The chromatograms didn’t yield any peaks for mobile phase and placebo when analyzed with the proposed HPLC method. No absorbance was found for blank/dilution fluid when analyzed spectrophotometrically using the proposed UV method. The results obtained were presented in table 10. The well-shaped peaks and the linearity of the results indicate that the proposed methods are selective and specific. A model chromatogram was illustrated in fig. 4.

Table 10: Selectivity and specificity of CPTB samples using proposed UV and HPLC methods

Mobile phase/Dilution liquid

Placebo

CPTB sample

Peak area/Absorbance

UV method

No absorbance

No absorbance

0.258

HPLC method

No peak

No peak

638125


Fig. 4: Typical chromatogram of CPTB


Table 11: Formulation analysis results

S. No.

Tablet name

Dose

Sample concentration

Sample estimated

% of drug estimated in tablet

1

CAPEGUARD (UV)

500 mg

1 mg/ml

0.99±0.0002 mg/ml

99.0 %

2

CAPEGUARD (HPLC)

500 mg

5 mg/ml

4.98±0.0001 mg/ml

99.7%

Determination of an active ingredient in bulk and in tablet dosage form (Formulation analysis)

Twenty solutions of CPTB were prepared using bulk drug and tablet dosage form (capegaurd). The samples were analyzed with both the proposed methods using the same experimental conditions and the drug content was found to be within the limits specified by I. P. The results obtained were presented in table 11.

F-test results for UV and HPLC method revealed that the Fcal value<Ftab value proving that null hypothesis is accepted. Hence it was proved that in both the methods, there is no significant difference between sample concentration and the sample estimated. The results also assured that both the proposed methods are selective for estimation of formulations.

CONCLUSION

A novel, precise, economical, accessible, reliable and reproducible method for estimation of CPTB in bulk and tablet dosage form using UV and HPLC methods were developed and were validated according to ICH guidelines. The wide range of linearity and use of readily available and economical mobile phase and dilution fluids establishes a further scope of promoting the proposed methods for estimation of capecitabine. The RSD values for all the validation parameters were found to be less than 1, indicating that the proposed UV and HPLC methods were trusts worthy. Both the methods have ample scope and application in industry for estimation of CPTB.

ACKNOWLEDGEMENT

The authors are grateful to the management of Vignan Pharmacy College for providing necessary facilities for the fulfillment of the current work.

CONFLICT OF INTERESTS

The authors express no conflict of interests

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How to site this article

  • K Pallavi, P. Srinivasa Babu, G Kishore Babu. Development and validation of UV spectrophotometric method and RP-HPLC method for estimation of capecitabine in bulk and tablet dosage forms. Int J Appl Pharm 2016;8(3):24-29


About this article

Title

DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD AND RP-HPLC METHOD FOR ESTIMATION OF CAPECITABINE IN BULK AND TABLET DOSAGE FORMS

Date

19-06-2016

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Journal

International Journal of Applied Pharmaceutics
Vol 8, Issue 3, 2016 Page: 24-29

Online ISSN

0975-7058

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Authors & Affiliations

K. Pallavi
Department of Pharmaceutics, Vignan Pharmacy College, Vadlamudi, Guntur (dt.), ap, 522213
India

P. Srinivasa Babu

G. Kishore Babu


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