INTRODUCTION:

Chemically Capecitabine is designated as pentyl [1- (3,4-dihydroxy-5-methyl- tetrahydrofuran-2-yl)- 5- fluoro-2-oxo-1H-pyrimidin- 4- yl] amino ethanoate. Capecitabine is an orally-administered chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers. Capecitabine is a prodrug that is enzymatically converted to fluorouracil (antimetabolite) in the tumor, where it inhibits DNA synthesis and slows growth of tumor¹. Capecitabine is a fluoropyrimidine carbonates with antineoplastic activity and it is in a class drugs known as anti-metabolites. Capecitabine is an orally administered chemotherapeutic agent used in treatment of metastatic breast and colorectal cancers^2,3,4. Capecitabine structure is shown in Figure1. Literature suggests few HPLC methods coupled with UV detection, fluorescence detection or mass spectrometry has been developed for the determination of Dimethyl sulfate in Capecitabine⁵

MATERIAL AND METHODS:

As per the literature survey there are no derivatization methods are available for the determination of dimethyl sulfate in capacitance^6,7. Impurities can have unwanted pharmacological or toxicological effects that seriously impact product quality and patient safety. According to ICH, an impurity in a drug substance is defined as any component of the new drug substance that is not the chemical entity defined as new drug substance^8,9. The identification threshold is the level at which an impurity must be structurally identified^10,11. The qualification threshold is the level at which the impurity in the drug product must be qualified for safety¹².

RESULTS AND DISCUSSION:

Derivatization:

Derivatization is the process by which a compound is chemically changed, producing a new compound that has properties more amenable to a particular analytical method. Derivatization in HPLC can be performed either before the chromatographic analysis (precolumn) or after the column separation and before detection (post column derivatization). The precolumn derivatization can be performed off-line in reaction vials.

Properties of derivatization reagent:

· The reagent should produce more than 95% complete derivatives.

· It should not cause any rearrangements or structural alterations of compounds during formation of the derivative.

· It should not contribute to loss of the sample during the reaction.

· It should produce a derivative that will not interact with the HPLC column.

· It should produce a derivative that is stable with respect to time.

Reaction between P-Nitro phenol with di methyl sulfate in presence of base is shown Figure 2.

Qualification of impurities:

Qualification is the process of acquiring and evaluating data that establishes the biological safety of an individual impurity or a given impurity profile at the levels specified^13,14. The applicant should provide a rationale for establishing impurity acceptance criteria that includes safety considerations. The level of any impurity present in a new drug substance that has been adequately tested in safety and/or clinical studies would be considered qualified.

Experimental section:

Samples and reagents:

The drug development samples of Capecitabine and Dimethyl sulfate were received from process development laboratory of Dr. Reddy’s Laboratories Ltd., Acetonitrile (HPLC grade) was purchased from Merck (India) Limited. Milli-Q grade water was obtained from the Milli-Q Plus water purification system used.

Instrumentation:

High performance liquid Chromatography (RP-HPLC) instrument used with Agilent Infinity 1260 series. Weighing of standards and test samples were carried out using analytical balance with Sartorius make and MSA 225S-100-DA model. The obtained chromatographic data were integrated through Empower-3 software.

Chromatographic Conditions:

Quantification was carried out on Kromasil C18, 250mm x 4.6mm x 3.5µm. Mobile phase-A was selected as 100% Milli Q water and Mobile phase –B was selected as 100% Acetonitrile. Diluent was selected as acetonitrile and water in the ration 65:35 (%v/v). Injection volume was 40 µL. Column flow maintained at 1.0mL/min with gradient program. Gradient program T/%B: 0/20,40/85,40.1/20 and 45/20. The temperature of column oven was maintained at 35°C. Based on UV spectra of DIS, wavelength for the detection was selected as 330nm. Injection volume was set as 40µL. Sample concentration was taken as 100.0mg/mL.

Standard and Sample Preparation¹⁵:

1.0% p-Nitro phenol stock solution:

Weigh and transfer about 100mg of sodium hydroxide pellets in to a 100ml volumetric flask, add 50ml of diluent, add 405mg of p-Nitro phenol and cool the solution to 10°C and dilute up to mark with diluent. Transfer 100µl of p-Nitrophenol stock solution (100%) in to a 10ml volumetric flask, dissolve and dilute up to mark with diluent.

Preparation of Dimethyl Sulphate (1.79ppm):

Transfer 20µl of DMS in to a 20ml volumetric flask, and made up to mark with 100% p-Nitrophenol stock solution, heat on water bath at 60°C for 60 minutes. Cool the solution to room temperature. Transfer 135µl of Dimethyl sulfate stock solution-1 in to a 10ml volumetric flask, dissolve and dilute up to mark with diluent. Transfer 100µl of Dimethyl sulfate stock solution-2 in to a 10ml volumetric flask, dilute up to mark with diluent.

Blank solution preparation:

Transferred 100µl of p-Nitro phenol solution in to a 100 ml volumetric flask, dilute up to mark with diluent. Heat on water bath at 60°C for 60 minutes. Cool the solution to room temperature.

Test sample Preparation:

Weighed accurately and transferred 1000mg of test sample in to a 10ml volumetric flask, add 100µl of 1.0 % p-Nitrophenol solution, dissolve and dilute up to mark with diluent. Heat on water bath at 60°C for 60 minutes. Cool the solution to room temperature. % RSD of 1.79 ppm of Dimethyl sulfate standard areas from six replicate standard injections was monitored as system suitability criteria.

Validation:

This Analytical method was validated to establish the suitability of the proposed method for the intended purpose. More specifically, it is a matter of establishing documented evidence providing a higher level of assurance with respect to the consistency of the method and results. It evaluates the product against defined specifications. The validation parameters viz., specificity, accuracy, precision, linearity, limit of detection, limit of quantitation, robustness, system suitability have to be evaluated as per the ICH guidelines^16,17.

Specificity¹⁸:

The specificity of an analytical method is the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. The basic criteria to show the specificity is no peak should be interfere at the retention time of peak of interest. Dimethyl sulfate is eluted at ~Retention time of ~21.0 min and there are no other peaks eluted at this particular retention time..

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

Limit of detection and Limit of quantification values for Dimethyl sulfate was established by preparing the known concentration solutions from their stock solutions that would give a signal to noise ratio of 3:1 and 10:1 respectively. The LOQ Precision was carried out by preparing six individual solutions at LOQ level. Calculated %RSD for the areas of Dimethyl sulfate. Accuracy at the Limit of Quantification level was performed by preparing three accuracy solutions of Capecitabine by spiking Dimethyl sulfate at LOQ level concentration. Calculated % recovery against the impurity spiked. Blank, LOD and LOQ and standard chromatograms are shown in Figures 4-7.

Linearity:

The linearity is one of the essential parameters of validation¹⁹, which gives the assurance on reported results by using the subject method at different concentration levels. Linearity was executed by preparing the solutions of Dimethyl sulfate at different concentration levels i.e., from LOQ to 0.15% against the target analyte concentration. Plotted the linearity graphs of peak areas versus concentration. These linearity curves give the information related to slope, intercept, correlation coefficients of regression and percent y-intercept. Linearity results calibration curves were shown in Figure 8.

Figure 8: Linearity graph of Dimethyl sulfate

Precision²⁰:

The precision was carried out by calculating the % RSD for the content of Dimethyl sulfate in each precision preparation. The precision solutions were prepared by spiking the Dimethyl sulfate impurity at specification level in to six different flasks. Each preparation was injected as per the developed method conditions. The same parameter was executed by changing the column, analyst and instrument in the same laboratory in order to establish the intermediate precision.

Accuracy²¹:

In order to carry out an accuracy parameter, solutions were prepared at three different concentrations i.e., 0.05, 0.10 and 0.15% w/w of analyte concentration. The % recovery for Dimethyl sulfate impurity was calculated as an amount of impurity obtained against an amount of impurity spiked

Solution stability:

Solution stability of the method was evaluated by injecting the capecitabine spiked with dimethyl sulfate standard at specification level at different time intervals. Solution stability was evaluated at 12 hrs, 24 h and 48 h and compared with initial results. These solution stability data are indicating that the solution stable up to 48 hrs

CONCLUSION:

A method for the quantification of Dimethyl sulfate by RP-HPLC in the Capecitabine API has been successfully developed and validated as per the ICH guidelines. As per the literature search there is no analytical method was available for the determination of Dimethyl sulfate. The validation results shown, that proposed RP-HPLC method was precise, accurate, linear and specific. Hence this method can be conveniently used to identify and quantify Dimethyl sulfate in regular Capecitabine test samples.

ACKNOWLEDGEMENT:

The first authors wish to thank the organization of Dr. Reddy’s Laboratories Ltd., for allowing this work to be published. Support extended by all the colleagues of Analytical Research and development department and Process Research and development department is thankfully acknowledged.

CONFLICT OF INTERESTS:

The authors declare that they have no conflict of interest.

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Received on 24.12.2020 Modified on 25.07.2021

Research J. Pharm. and Tech 2022; 15(10):4353-4358.

DOI: 10.52711/0974-360X.2022.00730