Development And Validation Of A Stability-Indicating Uplc Method For The Simultaneous Estimation Of Tipiracil And Trifluridine In Bulk And Pharmaceutical Dosage Forms

Gastric and colorectal cancers remain major causes of cancer-related morbidity and mortality worldwide, with advanced disease frequently associated with poor prognosis and limited therapeutic options after failure of standard treatment. The management of metastatic gastrointestinal malignancies has progressively evolved through the introduction of targeted agents and oral chemotherapeutic combinations; however, patients who progress after multiple treatment lines continue to represent a substantial therapeutic challenge. Consequently, the development of effective fluoropyrimidine-based strategies remains an important component of treatment for advanced gastrointestinal cancers (1,2).

Trifluridine/tipiracil (FTD/TPI; TAS-102)

is an orally administered antineoplastic combination developed to overcome some limitations associated with conventional fluoropyrimidine therapy. Trifluridine is a thymidine-based nucleoside analogue that becomes incorporated into DNA following intracellular phosphorylation, resulting in DNA dysfunction and inhibition of tumour cell proliferation. Its clinical utility is enhanced by tipiracil, a potent inhibitor of thymidine phosphorylase that prevents the rapid degradation of trifluridine and thereby increases its systemic exposure. The complementary actions of the two components provide sustained antitumour activity and form the pharmacological basis of the TAS-102 formulation (6–9).

Clinical and regulatory evaluations have established trifluridine/tipiracil as an important therapeutic option for patients with previously treated advanced or metastatic gastrointestinal malignancies. The combination has demonstrated clinical benefit in heavily pretreated patients with metastatic colorectal cancer and has subsequently been incorporated into treatment strategies for advanced gastric and gastroesophageal cancers. Its established clinical application and oral administration have increased the importance of reliable pharmaceutical quality-control procedures capable of accurately determining both active components in finished dosage forms (1,3–7).

The simultaneous quantitative determination of trifluridine and tipiracil presents an analytical challenge because both compounds possess distinct physicochemical and chromatographic characteristics. A suitable analytical procedure must therefore provide adequate selectivity and resolution while maintaining accuracy and precision in the presence of formulation excipients and potential degradation products. Stability-indicating methods are particularly important because degradation may occur during manufacturing, storage, transportation, and exposure to environmental stress. Forced degradation studies provide information regarding degradation behaviour and help demonstrate that an analytical procedure can distinguish the intact drug substances from their degradation products (20–22).

Several analytical procedures have been reported for the simultaneous determination of trifluridine and tipiracil. RP-HPLC methods have been developed for assay determination and stability assessment in bulk drug substances and combined pharmaceutical formulations, with reported methods demonstrating satisfactory linearity, precision, accuracy, and chromatographic separation (12–16). A selective LC–MS/MS/QTOF approach has also been reported for the separation and characterisation of trifluridine, tipiracil, and their degradation products, providing valuable information regarding degradation behaviour (11). Despite these developments, conventional HPLC procedures may involve comparatively longer analysis times and higher mobile-phase consumption. Furthermore, continued advances in pharmaceutical quality control require rapid and efficient analytical procedures capable of supporting high-throughput analysis.

Ultra-performance liquid chromatography (UPLC) provides enhanced chromatographic efficiency through the use of small particle-size stationary phases, enabling improved resolution, shorter analysis times, and lower solvent consumption compared with conventional HPLC. These characteristics make UPLC particularly attractive for routine pharmaceutical analysis and stability studies. A validated UPLC procedure can therefore provide an efficient analytical platform for simultaneous determination of trifluridine and tipiracil while maintaining the selectivity required for stability-indicating applications.

Analytical method validation is essential to establish the reliability and suitability of a procedure for its intended purpose. ICH Q2 guidelines recommend evaluation of parameters including specificity, linearity, accuracy, precision, and robustness, whereas ICH Q1A(R2) provides guidance concerning stability testing and degradation assessment of pharmaceutical products (17,18,21).

Therefore, the present study was undertaken to develop and validate a rapid, accurate, precise, robust, and stability-indicating UPLC method for the simultaneous determination of trifluridine and tipiracil in bulk drug substances and pharmaceutical dosage forms. The developed method was further evaluated through forced degradation studies to establish its ability to resolve the drug substances from their degradation products and to demonstrate its suitability for routine quality control and stability assessment of the combined formulation.

2. MATERIALS & METHODS

Chemicals and Reagents

Certified reference standards of trifluridine and tipiracil were obtained from a recognised pharmaceutical source. A marketed fixed-dose tablet formulation containing trifluridine and tipiracil was procured from the commercial market. HPLC-grade acetonitrile, methanol, and purified water were used for chromatographic analysis. Orthophosphoric acid of analytical-reagent grade was used for preparation of the aqueous mobile-phase component. All reagents and solvents were of suitable analytical or chromatographic grade and were used without further purification.

Instrumentation

Chromatographic analysis was performed using a UPLC system equipped with a binary solvent delivery system, autosampler, column oven, and photodiode-array (PDA) detector. Chromatographic data acquisition and processing were performed using the corresponding chromatography data system. An analytical balance, ultrasonic bath, calibrated volumetric glassware, pH meter, and membrane filtration unit were used during sample preparation and method development. Mobile phases and sample solutions were filtered through 0.22 μm membrane filters before chromatographic analysis.

Chromatographic Conditions

Chromatographic separation of trifluridine and tipiracil was achieved using a reversed-phase C18 UPLC column with suitable dimensions and sub-2 μm particle size. The aqueous phase consisted of 0.1% orthophosphoric acid, which was combined with acetonitrile as the organic modifier. The final mobile-phase composition, flow rate, column temperature, detection wavelength, injection volume, and total run time were optimised during method development to achieve satisfactory resolution, peak symmetry, and analysis time.

The developed chromatographic method was operated under isocratic elution conditions, with detection performed using a PDA detector at the selected analytical wavelength. Prior to sample analysis, the column was equilibrated with the mobile phase until stable baseline and reproducible system-suitability characteristics were obtained.

Preparation of Mobile Phase

The required volume of purified water was measured and acidified with orthophosphoric acid to prepare the aqueous mobile-phase component. The aqueous phase was mixed with acetonitrile in the optimised proportion and filtered through a 0.22 μm membrane filter. The mobile phase was subsequently degassed by sonication before use. Freshly prepared mobile phase was used for chromatographic analysis.

Preparation of Standard Stock Solutions

Accurately weighed quantities of trifluridine and tipiracil reference standards were separately transferred into suitable volumetric flasks. Each standard was dissolved in the selected diluent with the aid of sonication and diluted to volume to obtain individual stock solutions. Appropriate aliquots of the respective stock solutions were subsequently combined and diluted with the diluent to prepare a mixed standard solution containing both analytes at the desired working concentrations.

Preparation of Sample Solution

Twenty tablets were accurately weighed individually, and the average tablet weight was calculated. The tablets were finely powdered and an accurately weighed portion of the powder equivalent to the required label claim of trifluridine and tipiracil was transferred into a volumetric flask. A suitable quantity of diluent was added, followed by sonication to facilitate complete extraction of the active pharmaceutical ingredients. The solution was allowed to cool, diluted to volume with the diluent, and mixed thoroughly. An aliquot of the resulting solution was filtered through a 0.22 μm membrane filter and further diluted to obtain the final sample concentration suitable for UPLC analysis.

Method Development and Optimisation

Method development was initiated by evaluating different reversed-phase stationary phases, aqueous-organic mobile-phase compositions, flow rates, detection wavelengths, and column temperatures. The chromatographic conditions were systematically optimised based on retention behaviour, peak shape, theoretical plate count, tailing factor, resolution, baseline stability, and total analysis time. The final conditions were selected to provide reproducible separation of trifluridine and tipiracil within a short chromatographic run while maintaining adequate resolution from potential degradation products.

Method Validation

The developed UPLC method was validated in accordance with ICH Q2(R1) recommendations (17,18). The analytical characteristics evaluated included system suitability, specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), limit of quantitation (LOQ), solution stability, and stability-indicating capability.

System Suitability

System suitability was assessed by injecting the mixed standard solution repeatedly under the optimised chromatographic conditions. Retention time, peak area, theoretical plate count, tailing factor, and chromatographic resolution were evaluated. The percentage relative standard deviation (%RSD) of replicate injections was calculated to verify the reproducibility of the chromatographic system.

Specificity

Specificity was evaluated by separately analysing the diluent, placebo, standard solution, sample solution, and stressed sample solutions. The chromatograms were examined for any interference at the retention times of trifluridine and tipiracil. PDA peak-purity analysis was additionally employed to establish the spectral homogeneity of the analyte peaks.

Linearity

Linearity was assessed by preparing a series of standard solutions covering the selected concentration ranges of trifluridine and tipiracil. Each concentration was analysed under the optimised chromatographic conditions. Calibration curves were constructed by plotting the corresponding peak areas against nominal concentrations, and linear regression analysis was performed to obtain the slope, intercept, and correlation coefficient.

Accuracy

Accuracy was evaluated using the standard-addition technique at three concentration levels corresponding to 50%, 100%, and 150% of the nominal concentration. Each level was analysed in triplicate. Percentage recovery and %RSD were calculated to establish the closeness of the measured values to the theoretical concentrations.

Precision

Precision was investigated as repeatability and intermediate precision. Repeatability was determined by analysing multiple independently prepared sample solutions under identical conditions on the same day. Intermediate precision was evaluated on different days and, where applicable, by different analysts. The results were expressed as %RSD of the assay values.

Limit of Detection and Limit of Quantitation

LOD and LOQ were determined from the standard deviation of the response and the slope of the calibration curve using the following equations:

LOD = 3.3σ/S

LOQ = 10σ/S

where σ represents the standard deviation of the response and S represents the slope of the calibration curve.

Robustness

Robustness was assessed by introducing deliberate but minor variations in selected chromatographic parameters, including flow rate, mobile-phase composition, detection wavelength, and column temperature. The effect of these changes on retention time, peak shape, resolution, and assay results was evaluated to determine the resilience of the method under normal laboratory variations.

Solution Stability

The stability of standard and sample solutions was investigated by storing the prepared solutions under laboratory conditions for a predetermined period. The solutions were analysed at selected time intervals and the results were compared with those obtained from freshly prepared solutions. Solution stability was considered acceptable when no significant change in assay response or chromatographic performance was observed.

Forced Degradation Studies

Forced degradation studies were performed to establish the stability-indicating capability of the proposed UPLC method in accordance with the principles of ICH Q1A(R2) (21). The drug product was subjected to different stress conditions, including acidic hydrolysis, alkaline hydrolysis, oxidation, thermal degradation, photolytic degradation, and neutral hydrolysis. Appropriate stress conditions and exposure times were selected to produce measurable degradation without complete loss of the parent compounds.

Following stress treatment, acidic and alkaline samples were appropriately neutralised where required, diluted with the selected diluent, filtered, and analysed using the optimised UPLC method. The chromatographic profiles were compared with those of unstressed samples. The ability of the method to separate trifluridine and tipiracil from their respective degradation products was assessed based on retention behaviour, resolution, peak purity, and overall chromatographic performance. The results were used to confirm that the proposed method was capable of selectively quantifying the intact drug substances in the presence of their degradation products.

3. RESULTS AND DISCUSSION

Method Development

Table 1: Preliminary Trials for UPLC Method Development

Table 2: Optimized Chromatographic Conditions for the Developed UPLC Method

Method Validation:

System Suitability

Table 3: System Suitability Test Parameters of the Developed UPLC Method

Linearity:

Table 4: Linearity Studies of the Proposed UPLC Method

Table 5: Linear Regression Data

Precision:

Table 6: Results of Repeatability Studies (Intra-Day Precision)

Table 7: Results of Intermediate Precision (Inter-Day Precision)

Accuracy (% Recovery):

Table 8: Accuracy Studies at 50% Recovery Level

Table 9: Accuracy Studies at 100% Recovery Level

Table 10: Accuracy Studies at 150% Recovery Level

Table 11: Summary of Accuracy (% Recovery) Studies

Robustness:

Table 12: Robustness Study by Variation in Flow Rate

Table 13: Robustness Study by Variation in Mobile Phase Composition

Table 14: Robustness Study by Variation in Detection Wavelength

Table 15: Robustness Study by Variation in Column Temperature

Table 16: Summary of Robustness Studies

Specificity:

Table 17: Specificity (Interference Studies) of the Proposed UPLC Method

Acceptance Criteria

LOD & LOQ

Table 18: LOD & LOQ

Assay of Pharmaceutical Formulation

Table 19: Assay of Pharmaceutical Formulation

Acceptance Criteria

Forced degradation studies:

Table 20: Forced Degradation Studies