INTRODUCTION:

Capecitabine is chemically pentyl [1-(3,4-dihydroxy-5-methyl-tetrahydrofuran-2-yl)-5-fluoro-2-oxo-1H-pyrimidin-4-yl]aminomethanoate an orally chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers and shown in Fig 1. It is a prodrug, that is enzymatically converted to 5-fluorouracil in the tumor, where it inhibits DNA synthesis and slows growth of tumor tissue.^1,2

Fig. 1. Structure of capecitabine

The activation of capecitabine follows a pathway with three enzymatic steps and two intermediary metabolites, 5'-deoxy-5-fluorocytidine (5'-DFCR) and 5'-deoxy-5-fluorouridine (5'-DFUR) to form 5-fluorouracil. Literature survey reveals few UV³, HPLC^4-8 and LC-MS^9,10 for the determination of capecitabine in combination with other metabolites in biological fluids and one in formulation. Proposed both UV and HPLC method for the estimation of capecitabine in pharmaceutical dosage form is simple, specific and fast. Till date there is no method for the single solvent UV as well as the HPLC. The present aims was both the methods used as a same solvent, less time consume very sensitive, selective and very short time isocratic RP HPLC method for the estimation of capecitabine in bulk as well as tablet dosage form.

MATERIALS AND METHODS:

Capecitabine was a gift sample from NATCO Laboratories Ltd, Hyderabad, India. HPLC grade acetonitrile, water and orthophosphoric acid were procured from Merck Pharmaceuticals Private Ltd., Mumbai, India.

Instrumentation:

Double beam UV-Visible Spectrophotometer (Shimadzu-1800) connected to a computer loaded with Shimadzu UV Probe 2.33 software.

Selection of solvent:

From the drug profile of capecitabine, initially water was selected as a solvent, precipitate was occurred and then later acetonitrile was found to be a solvent to produce a clear solution. But to developed the method acetonitrile: water (50:50) to produce a common solvent for both the methods.

Selection of wavelength:

10µg/ml of capecitabine were prepared in acetonitrile: water (50:50) solvent. The resulting solutions were scanned from 190 to 400 nm in UV-Visible spectrophotometer. The optimal response for two of them was obtained at 242 nm. Hence the complete method was processed at the wavelength of 242 nm.

Preparation of standard stock solution:

Standard stock solution of capecitabine (1 mg/ml) was prepared by transferring 10 mg of capecitabine into a 10 ml volumetric flask separately and add 4mL of acetonitrile: water (50:50) v/v. It was then sonicated for 15 minutes and solution was diluted up to the volume by a solvent. From these, further dilutions were made using acetonitrile: water (50:50) v/v to produce solution of capecitabine (10µg/ml).

Preparation of sample solution:

Twenty tablets of contents were weighed and the average weight was determined. They were crushed in to fine powder with glass mortar. The quantity of powder equivalent to 50mg capecitabine was transferred into a 10 ml clean dry volumetric flask separately and acetonitrile: water (50:50) v/v was used as solvent, added to it and was shaken by mechanical stirrer and sonicated for about 30 minutes by shaking at intervals of five minutes and it was diluted up to the mark with diluent to give a concentration of 5000µg/ml for capecitabine the solution was filtered through the Whatman filter paper. Further diluted with upto 100µg/ml.

Validation of UV spectroscopic method:

As per the International Conference on Harmonization (ICH) guidelines, the method validation parameters like linearity, precision, accuracy, limit of detection, limit of quantitation, specificity and robustness were experimentally determined and the method was validated.^11,12

Selection of analytical concentration ranges:

From the standard stock solution of capecitabne (100µg/ml), appropriate aliquots of 1ml, 2ml, 3ml, 4ml, 5ml and 6ml was pipette out and transferred into a 10ml volumetric flasks and dilutions were made with acetonitrile: water (50:50) v/v to obtain working standard solutions with concentration ranging from 1 to 6µg/ml.

Linearity:

The linearity of analytical method is its ability to elicit test results that are directly proportional to the concentration of analyte in the sample within the range. The range of the analytical method is the interval between the upper and lower levels that have been demonstrated to be determined within a suitable level of precision, accuracy and linearity. Appropriate aliquots from standard capecitabine stock solutions were transferred into different volumetric flasks of 10ml capacity. The volume was adjusted to the mark to obtain concentrations of 1, 2, 3, 4, 5, 6µg/ml. Absorbance spectra of each solution against acetonitrile: water (50:50)v/v as blank were measured at 242nm.

Precision:

The precision of an analytical method is the degree of agreement among individual test results, when the method is applied repeatedly to multiple sampling of homogeneous samples. It provides an indication of random error results and is expressed as relative standard deviation (%RSD).

Repeatability studies:

Repeatability expresses the analytical variability under the same operating conditions over a short interval of time. At least six determinations at 100% test concentration should be performed. The repeatability studies were carried out by taking 4µg/ml as the test concentration and repeating it for six times.

Intermediate precision:

Intra-day and Inter-day precision:

Variation of results within the same day (intra-day), variation of results between days (inter-day) were analysed. Intra-day precision was determined by analyzing for 6 times on the same day at 242nm. Inter-day precision was determined by analyzing 3 concentrations of on the preceding day at 242nm and %RSD was calculated.

Accuracy:

Accuracy is the closeness of the results obtained by the method to the true value. Recovery studies were carried out at 50%, 100% and 150% by adding known amount of standard drug solution of 3,4,5 µg/ml to the sample solution whose concentration is maintained constant at 2µg/ml. The %recovery was calculated.

Ruggedness:

The solutions were prepared and analysed with change in the analytical conditions like different instrument and different analyst. Standard concentrations of 4µg/ml were used to carry out the ruggedness studies.

Limit of detection (LOD) and Limit of quantification (LOQ):

The sensitivity of the proposed method for the measurement of capecitabine was estimated in terms of Limit of detection (LOD) and Limit of quantification (LOQ). The LOD and LOQ were calculated by using the slope and SD of response (intercept).

Instrumentation:

Reverse phase-High performance liquid chromatography (Agilent) equipped with VW detector. The software used is EZ Chrome and the column employed is Eclipse C₁₈ (100 mm × 4.6 mm, 3.5 μm particle size).

Selection of mobile phase:

Preparation of mobile phase:

Take HPLC water 50 ml (50%) and 50ml Acetonitrile (50%) HPLC into a 100 ml volumetric flask to this add (0.1%v/v) of ortho phosphoric acid was mixed and degassed in ultrasonic water bath for 5 minutes and was filtered through 0.45 µ filter under vacuum filtration.

Selection of flow rate:

As increase in the flow rate results in decrease in the retention time. Hence sufficient flow rate of 0.8 mLmin^-1 was chosen to avoid overlap between the peaks.

Preparation of Standard Stock Solution:

Standard stock solution of capecitabine (1 mg/ml) was prepared by transferring into a 10 ml volumetric flask containing 4mL of acetonitrile: water water containing 0.1% phosphoric acid v/v (50:50) diluent. It was then sonicated for 15 minutes and solution was diluted up to the volume by acetonitrile: water (50:50). From these, further dilutions were made using acetonitrile: water containing 0.1% phosphoric acid (50:50) to produce solution of capecitabine (10µg/ml).

Preparation of sample stock solution:

About 10mg of capecitabine samples was weighed in to volumetric flask, it was dissolved with diluents acetonitrile: water (50:50) and the volume was made up to the mark

Specificity:

The term specific generally refers to a method that produces a response for a single analyte only while the term selectivity refers to a method that provides responses for a number of chemical entities that may or may not be distinguished from each other. The analyte was assessed in the presence of components and it was found that there was no interaction with the analyte.

Linearity and range:

Each level solution was injected into the chromatographic system and the peak area was measured. A graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) was plotted and the correlation coefficient was calculated. The linearity of the method was demonstrated over the concentration range of capecitabine 1-6µg / ml. Aliquots of six levels were prepared from sample solution.

Precision:

Repeatability:

Repeatability expresses the analytical variability under the same operating conditions over a short interval of time. The repeatability studies were carried out by taking 4µg/ml for capecitabine as 100% test concentration and repeating it for six times.\

Intermediate precision:

Intra day precision and inter day precision:

Variation of results within the same day (intra-day) and variation of results between days (inter-day) were analysed. Intra-day precision was determined by analyzing single concentration of capecitabine for 6 times on the same day at 242nm. Inter-day precision was determined by analyzing single concentrations of the preceding day at 242nm and %RSD was calculated.

Accuracy:

Accuracy is the closeness of the results obtained by the method to the true value. Recovery studies were carried out at 50%, 100% and 150% by adding known amount of standard drug solution of i.e. (3,4 and 5 µg/ml) to the sample solution. The %recovery was calculated and reported.

Ruggedness:

The solutions were prepared and analysed with change in the analytical conditions like different instrument and different analyst. Standard concentrations of 3µg/ml were carried used to carry out the ruggedness studies.

Robustness:

Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage. It is carried out by changing the flow rate of mobile phase from 0.8 to 1.2 mLmin^-1,changing the detection wavelength from 240 to 242nm.

System suitability testing (SST):

System suitability tests are an integral part of chromatographic method. They were used to verify that the reproducibility of the chromatographic system is adequate for the analysis. According to the USP, system suitability tests are an integral part of chromatographic methods. These tests are used to verify that the resolution and reproducibility of the system are adequate for the analysis to be performed. System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples constitute an integral system that can be evaluated as a whole. The purpose of the system suitability test is to ensure that the complete testing system (including instrument, reagents, columns, analysts) is suitable for the intended application. System suitability test parameters are: Plate number or number of theoretical plates (n) ratio) k, The selectivity or Separation Factor (relative retention) α. These are measured on a peak or peaks of known retention time and peak width.

Limits of detection and limits of quantification:

The parameters LOD and LOQ were determined on the basis of response and slope of the regression equation.

The flow rate was varied at 0.9 mLmin^-1 to 0.7 mLmin^-1:

Standard solution of capecitabine was analysed at 0.9 and 0.7mL min-1 i.e. at ± 0.8unit of optimized flow rate (1mL/min).

The detection wavelength was varied at 240 to 244nm:

Standard solution of capecitabine was analysed at 240 and 244nm i.e. at ± 1unit of optimized wavelength (242nm).

Selectivity studies:

The solutions were prepared and analysed with change in the analytical conditions like different instrument and different analyst. Standard concentrations of capecitabine 1µg/ml were carried used to carry out the ruggedness studies.

Assay studies:

Assay studies were carried out by weighing twenty tablets of Capecitabine formulation and powdered. The powder equivalent to 10mg was taken and the solution equivalent to 1000µg/ml was prepared and was used for further dilutions.

RESULTS:

UV Spectrophotometry:

Selection of wavelength:

The standard stock solution of capecitabine of 10µg/ml concentration was scanned from 200-400nm and the absorption spectra were recorded at 242nm wavelength in UV spectrophotometer which is presented in Fig.2.

Fig. 2. Absorption spectrum of capecitabine

Linearity:

The linearity was found in the concentration range of capecitabine i.e. 1-6 µg/ml for the developed UV spectrscopy method. The X-axis is concentration and the Y-axis is absorbance. The correlation coefficient was found to be 0.9991 and the regression equation was found to be Y=0.1672x + 0.0246 for capecitabine and the graph is shown in Fig 3.

Fig. 3. UV Calibration curve for capecitabine

Precision:

The %RSD value for repeatability, intraday and interday is 0.165, 0.587-1.882, 0.092-1.903, for capecitabine the results shown in table 1 and 2 which indicate that they meet the acceptance criteria and hence the method is precise.

Table 1 Repeatability studies of capecitabine

Concentration (µg/ml)	Absorbance (242nm)	Mean absorbance ± S.D	%RSD
4	0.626	0.625±0.583	0.02
4	0.624
4	0.626
4	0.627
4	0.626
4	0.625

Table 2 Intraday precision and interday precision of capecitabine

Concentration (µg/ml)	Intraday precision			Interday precision
	Absorbance (242nm)	Absorbance mean± S.D	%RSD	Absorbance (242nm)	Absorbance mean± S.D	%RSD
2	0.310	0.306± 0.005	1.882	0.310	0.303±0.005	1.903
	0.300			0.300
	0.310			0.300
4	0.627	0.626± 0.001	1.082	0.627	0.626±0.0005	0.092
	0.627			0.626
	0.625			0.626
6	0.990	0.983± 0.005	0.583	0.990	0.983±0.011	1.174
	0.980			0.970
	0.980			0.990

Table 3 Accuracy studies of capecitabine

Spiked level (%)	Formulation Conc (µg/ml)	Pure Drug Conc (µg/ml)	Amount found	% Recovery	% Mean recovery ±SD	%RSD
50	2	1	2.949	98.3	99.2±1.398	1.409
	2	1	2.955	98.5
	2	1	3.025	100.8
100	2	2	4.025	100.6	100.3±1.150	1.146
	2	2	3.961	99.0
	2	2	4.051	101.2
150	2	3	4.950	99.0	99.6±0.621	0.623
	2	3	5.012	100.0
	2	3	4.987	99.7

Table 4 UV Ruggedness studies of capecitabine

Parameter	Concentration (µg/ml)	Absorbance (242nm)	Absorbance (mean± S.D)	%RSD
Different instrument	4	0.627	0.626±0.0005	0.092
	4	0.626
	4	0.626
Different analyst	4	0.625	0.626±0.001	0.159
	4	0.626
	4	0.627

Table 8 HPLC Intermediate Precision studies of capecitabine

Conc	Intraday Precision				Interday Precision
Conc	Peak Area	Mean peak area	RSD	R_t	Peak Area	Mean peak area	RSD	R_t
1	5149782	5151041± 20905.4	0.40	1.775	5131783	5146409± 11313.1	0.21	1.775
	5141782			1.610	5130783			1.677
	5140782			1.810	5131783			1.810

Table 9 HPLC Accuracy studies of capecitabine

Spiked level (%)	Formulation Conc (µg/ml)	Pure Drug Conc(µg/ml)	Amount found	% Recovery	% Mean recovery ±SD	%RSD
50	0.5	0.75	0.7465	99.53	99.48±0.2759	0.2773
	0.5	0.75	0.7480	99.73
	0.5	0.75	0.7439	98.18
100	0.5	1	0.9950	99.50	99.653±0.134	0.135
	0.5	1	0.9976	99.76
	0.5	1	0.9969	99.69
150	0.5	1.25	1.2372	98.98	98.897±0.244	0.247
	0.5	1.25	1.2386	99.98
	0.5	1.25	1.232	98.62

Robustness:

The changing the flow rate of mobile phase from 0.8 to 1.2 mL min^-1,changing the detection wavelength from 240 to 242nm and results are mentioned table 10.

Table 10. HPLC Robustness studies of capecitabine

Parameter	R_t	Area	Height	Plate count
Flow Rate Less Flow	2.253	2558248	234950	4162
Flow Rate More flow	1.680	2084296	225397	3921.4
Wavelength 244	1.693	4026730	211957	4457.1
Wavelength 240	1.693	4270708	245935	3712.3

Ruggedness:

To determine ruggedness, two different analysts performed assay of the drug in similar operational and environmental conditions using developed method. Standard solution of capecitabine was analysed by analyst 1 and analyst mentioned table 11.

Table 11.HPLC Ruggedness studies of capecitabine

Para meter	Concentration (µg/ml)	Area	Area Mean± S.D	% RSD
Different analyst	1	5144782	5151041± 20905.04	0.4058
	1	5133981
	1	5174361

System suitability parameters:

Parameters such as a number of theoretical plates (N), tailing factor and retention time, resolution were calculated and mentioned in the table 12.

Table 12. System suitability studies of capecitabine

Injection	R_t	Peak Area	USP Plate count	Asymmetric factor
1	1.677	5134732	4012	1.5
2	1.677	5118491	4025	1.2
3	1.677	5089132	4137	1.5

Application to pharmaceutical dosage form:

Assay studies were carried out by taking twenty tablets of capecitabine formulation which were weighed and powdered. The powder equivalent to 10mg was taken and the solution equivalent to 1000µg/ml The results show that the %purity was found to be 99.33%for capecitabine and mentioned in table 13.

Table 13. HPLC Assay studies of capecitabine

Formulation	Label claim	% Purity	Mean peak± S.D	%RSD
Xeloda 500mg	500mg	99.33	51411823±112.34	0.4519

Limit of detection (LOD) and Limit of quantification (LOQ):

The mean slope value and the SD of response were obtained from the calibration curve and LOD and LOQ were shown in 0.047 and 0.1424.

DISCUSSION:

The optical characteristics such as Beer’s law limits molar absorptivity, LOD and LOQ in each method were calculate and the results are displayed e regression equation like slope(b), intercept(a) and correlation coefficient(R²) using the method of least squares were calculated The results show that the methods are reasonably precise, LOD and LOQ in each method were calculate and results are shown in table 14. The objective of both the methods was to develop new analytical methods for the determination of capecitabine to validate the methods according to the ICH guidelines The developed UV spectrophotometric and RP-HPLC method was found to be rapid, simple, precise, accurate and economic for routine estimation of capecitabine in pure and pharmaceutical dosage forms.

Table 14.Summary of UV and HPLC validation parameters for capecitabine

Parameter	Capecitabine	Capecitabine
max (nm)	242nm	242nm
Linearity range µg/ml	1-65µg/ml	0.25-1.5µg/ml
Correlation coefficient (r²)	0.999	0.999
Regression equation (y=mx+c)	Y=-0.024x-0.167	y=5E+06x+ 46652
Slope (m)	-0.024x	5E+06x
Intercept(c)	0.167	46652
Accuracy	98.3-101.2%	98.18-99.98
Precision (%RSD)	0.165	0.49
LOD	0.103	0.047
LOQ	0.312	0.1424

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6. K. Srinivasu, J Venkateswara Rao, N. Appala Raju and K. Mukkanti. RP-HPLC method for the estimation of capecitabine in tablet dosage forms. Asian Journal of Chemistry 22(4);2010: 3255-3259.

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Received on 16.04.2018 Modified on 25.04.2018

Asian J. Research Chem. 2018; 11(4):731-738.

DOI: 10.5958/0974-4150.2018.00129.3

Trials	Mobile phase	Observation	Remarks
1	Methanol : Water (70:30 v/v)	Broad peak appearance	Not satisfactory
2	Acetonitrile : Water (80:50 v/v)	Negative Extra peak with tailing	Not satisfactory
3	Methanol : Acetatonitrile: water (50:30: 20 v/v/v)	Sharp peak with tailing	Not satisfactory
4	Methanol : Acetate buffer: water (50:40: 10 v/v/v)	Sharp peak with small fronting	Not satisfactory
5	Acetonitrile: Water (70:30 v/v)	Sharp peak with extra peak	Not satisfactory
6	Acetonitrile: Water (50:50 v/v)	Sharp peak appear	Satisfactory

Concentration (µg/ml)	Absorbance	Area mean± S.D(n=6)	%RSD
1	5147832	5169153±25816.7	0.49
1	5147832
1	5147832
1	5147832
1	5147832
1	5147832

Drug name	Formulation	Label claim	Amount found	%Purity	Mean absorbance± S.D	%RSD
Capecitabine	Xeloda	500mg	495mg	99%	0.308±0.004	1.53