RP- HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF TIPIRACIL AND TRIFLURIDINE IN TABLET DOSAGE FORM

Jutta Swathi. et al. © 2012, JPR. All Rights Reserved Jo Vol. 14 RPHPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF TIPIRACIL AND TRIFLURIDINE IN TABLET DOSAGE FORM P. Sri Harsha Depart ment of Pharmaceutical Analysis, R eceived on: A simple, Accurate, precise method was developed for the simultaneous estimation of the Tipiracil and Trifluridinein Tablet dosage form. Chromatogram was run through C18Hypertsil 5µ, 250mm×4.6mm column u phosphoric acid in water: acetonitrile (pH 4 using sodium hydroxide) as mobile phase. The retention time for Lamivudine and Tipiracil and Trifluridinewere 2.1 min and 4.2 min respectively. The method is linear over a concentration range o for Tipiracil and 50 to 300 µg/ml for Trifluridine. The method wwas precise with % RSD within the acceptance limits.The metho validated for system suitability, accuracy, precision, linearity and ruggedness. The system suitability p hence it was concluded that the method was suitable to perform the assay. Keywords: Tipiracil, Trifliridine, RPHPLC, Simultaneous estimation, Method validation INTRODUCTION C olorectal cancer (CRC) remains a significant global health burden, demanding the development of effective therapeutic strategies. The combination of Tipiraciland Trifluridine(TFD) has emerged as a promising treatment option for patients with metastatic CRC. Tipiracil, acting as a thymidine phosphorylase inhibitor, potentiates the antitumor activity of TFD, a nucleoside analog that disrup ts DNA synthesis in cancer cells. While both Tipiracil and TFD play crucial roles in this therapeutic approach, there is a paucity of robust and selective methods for their simultaneous quantification in commercially available tablet dosage forms. Existin g literature primarily focuses on individual drug analysis or methods applicable to other drug combinations. This scarcity of readily available analytical methods for the TipiracilTFD combination presents a Corresponding author: Jutta Swathi Department of Pharmaceutical Analysis Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Tarnaka, Secunderabad, Telangana, India Email: [email protected] DOI: J Pharm Res, 2025; 14(06): 27 https://jprinfo.com/ Jo urnal of Pharma Research Available online through www.jprinfo.com 14 Issues 06, 2025 ISSN: 2319-5622 Original Article HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF TIPIRACIL AND TRIFLURIDINE IN TABLET DOSAGE FORM P. Sri Harsha 1*, P. Vivek Sagar1, S. HemaLatha1, Jutta Swathi1 ment of Pharmaceutical Analysis, Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Tarnaka, Secunderabad eceived on: 01-10-2025 Accepted on: 03-11-2025 ABSTRACT simple, Accurate, precise method was developed for the simultaneous estimation of the Tipiracil and Trifluridinein Tablet dosage form. Chromatogram was run through C18Hypertsil 5µ, 250mm×4.6mm column u phosphoric acid in water: acetonitrile (pH 4 using sodium hydroxide) as mobile phase. The retention time for Lamivudine and Tipiracil and Trifluridinewere 2.1 min and 4.2 min respectively. The method is linear over a concentration range o for Tipiracil and 50 to 300 µg/ml for Trifluridine. The method wwas precise with % RSD within the acceptance limits.The metho validated for system suitability, accuracy, precision, linearity and ruggedness. The system suitability p arameters were within limit, hence it was concluded that the method was suitable to perform the assay. . HPLC, Simultaneous estimation, Method validation . olorectal cancer (CRC) remains a significant global health burden, demanding the development of effective combination of Tipiraciland Trifluridine(TFD) has emerged as a promising treatment option for patients with metastatic CRC. Tipiracil, acting as a thymidine phosphorylase inhibitor, potentiates the antitumor activity of ts DNA synthesis in cancer Tipiracil and TFD play crucial roles in this therapeutic approach, there is a paucity of robust and selective methods for their simultaneous quantification in commercially g literature primarily focuses on individual drug analysis or methods applicable to other drug combinations. This scarcity of readily available TFD combination presents a Sarojini Naidu Vanita Pharmacy MahaVidyalaya, challenge for pharmaceutical quality control and potentially hinders optimal patient care. This present study aims to bridge this critical analytical gap by developing and validating a Reversed Liquid Chromatography (RPHPLC) method for the simultaneous estimation of Tipiracil and TFD in tablets. This optimized method offers s everal key advantages like enhanced efficiency by reducing analysis time and laboratory resource consumption compared to separate methods for each drug. The method was meticulously designed to achieve chromatographic separation of Tipiraciland TFD from pot degradation products and common tablet excipients, ensuring accurate and reliable quantification.The method was optimized for short analysis times and user routine application in quality control laboratories for the tipiracil-TFD combination product. 2. MATERIALS AND METHODS • Tipiracil and Trifluridinepure drugs (API) received as gift sample from NATCO pharma ltd.Marketed tablets of Tipiracil and Trifluridine (Lonsurf) was purchased from Indiamart Distilled water, Acetonitrile, Phosphate buffer, Methanol, J Pharm Res, 2025; 14(06): 27 -32 HPLC METHOD DEVELOPMENT AND VALIDATION FOR SIMULTANEOUS ESTIMATION OF Sarojini Naidu Vanita Pharmacy MahaVidyalaya, Tarnaka, Secunderabad simple, Accurate, precise method was developed for the simultaneous estimation of the Tipiracil and Trifluridinein Tablet dosage form. Chromatogram was run through C18Hypertsil 5µ, 250mm×4.6mm column u sing 0.1% ortho phosphoric acid in water: acetonitrile (pH 4 using sodium hydroxide) as mobile phase. The retention time for Lamivudine and Tipiracil and Trifluridinewere 2.1 min and 4.2 min respectively. The method is linear over a concentration range o f 22.5 – 135 µg/ml for Tipiracil and 50 to 300 µg/ml for Trifluridine. The method wwas precise with % RSD within the acceptance limits.The metho d was arameters were within limit, challenge for pharmaceutical quality control and potentially present study aims to bridge this critical analytical gap by developing and validating a Reversed -Phase High-Performance HPLC) method for the simultaneous estimation of Tipiracil and TFD in tablets. This optimized everal key advantages like enhanced efficiency by reducing analysis time and laboratory resource consumption compared to separate methods for each drug. The method was meticulously designed to achieve chromatographic separation of Tipiraciland TFD from pot ential degradation products and common tablet excipients, ensuring accurate and reliable quantification.The method was optimized for short analysis times and user -friendliness, facilitating its routine application in quality control laboratories for the Tipiracil and Trifluridinepure drugs (API) received as gift sample from NATCO pharma ltd.Marketed tablets of Tipiracil and Trifluridine (Lonsurf) was purchased from Indiamart Distilled water, Acetonitrile, Phosphate buffer, Methanol, Jutta Swathi. et al. J Pharm Res, 2025; 14(06): 27-32 © 2012, JPR. All Rights Reserved https://jprinfo.com/ Potassium dihydrogenortho phosphate buffer, Ortho-phosphoric acid. Alltheabovechemicals andsolvents are from Rankem 2.1. Solutions: 2.1.1. Preparation of Standard solutions: Twenty milligrams (20 mg) of trifluridine and 9 milligrams (mg) of tipiracil were accurately weighed. The weighed substances were then transferred to a 10 mL volumetric flask. Seven and one-half milliliters (7.5 mL) of diluent were added to the flask. The solution was sonicated for 10 minutes using a laboratory or handheld sonicator. Following sonication, the volume of the solution in the flask was brought to 10 mL with diluent. This flask was labeled as the "standard stock solution". Next, 1.0 mL of the standard stock solution was pipetted into a new 10 mL volumetric flask. This solution was then diluted to 10 mL with diluent and labeled as the "30 µg/mL solution". Likewise, 1.5 mL of the standard stock solution was pipetted into another new 10 mL volumetric flask. This solution was also diluted to 10 mL with diluent and labeled as the "13.5 µg/mL solution". 2.1.2. Samples Preparation A sample solution containing trifluridine and tipiracil can be prepared from tablets following a defined procedure. Ten tablets are first weighed to determine the average weight of a single tablet. A portion of the crushed tablets, equivalent to the weight of one tablet, is then transferred to a 10 mL volumetric flask. After adding a few milliliters of diluent, the mixture is sonicated for 25 minutes to ensure proper extraction. The volume of the solution is then brought to 10 mL with the same diluent used previously. Finally, the solution is passed through HPLC filters to remove any unwanted particles. This process yields a sample solution with a final concentration of 2000 µg/mL trifluridine and 900 µg/mL tipiracil. 2.1.3. Cc standards; A stock solution of Trifluridine and Tipiracil was prepared by accurately weighing 20 mg of Trifluridine and 9 mg of Tipiracil. These weighed substances were then transferred to a 10 mL volumetric flask. Subsequently, 7.5 mL of diluent was added to the flask, and the solution was sonicated for 10 minutes. Following sonication, the volume of the solution in the flask was brought to 10 mL with diluent. Next, aliquots of appropriate volume were pipetted from the stock solution into separate 10 mL volumetric flasks. The aliquots were chosen to achieve concentrations ranging from 50 to 300 µg/mL for trifluridine and 22.5 to 135 µg/mL for tipiracil within these flasks. Finally, the volume of the solutions in each volumetric flask was made up to 10 mL with diluent. These resulting solutions served as the calibration curve standards. 2.2. Diluent:Mobile phase is used as diluent. 2.3. Chromatographic conditions: The new HPLC method for estimation of Trifluridine and 9 mg Tipiracil was developed and validated using a Hypersil ODS C18 column (150 x 4.6mm 3.5mm). The mobile phase for this method was prepared by mixing 0.1% ortho phosphoric acid in water (adjusted to pH 4) and acetonitrile in a ratio of 65:35. Isocratic elution mode with a flow rate of 1.0 mL/min was employed for separation, and the eluent was monitored at a wavelength of 292 nm. 2.4. System suitability: In order to assess system suitability, standard solutions of Trifluridine and Tipiracil were prepared and injected six times. From these injections, several parameters were evaluated including peak tailing, resolution, and USP plate count. The acceptance criteria for system suitability dictated that the relative standard deviation (RSD) for the peak area of both drugs across the six replicate injections should not exceed 2%. Additionally, the tailing factor for both Trifluridine and Tipiracil should be less than or equal to 2. 2.5. Method validation The method validation was performed in accordance with ICH guidelines 2.5.1. Linearity Each concentration level of the calibration standards was injected into the chromatographic system, and the corresponding peak area for each drug was measured. A calibration curve was then constructed by plotting peak area on the y-axis versus concentration on the x-axis. The correlation coefficient of this calibration curve was subsequently calculated 2.5.2. Accuracy Each concentration level of the calibration standards was injected into the chromatographic system, and the corresponding peak area for each drug was measured. A calibration curve was then constructed by plotting peak area on the y-axis versus concentration on the x-axis. The correlation coefficient of this calibration curve was subsequently calculated 2.5.3. Precision The precision of an analytical procedure is defined by the closeness of agreement between a series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed conditions. It reflects the degree of repeatability or reproducibility of the analytical method. Typically, precision is expressed through variance, standard deviation, or coefficient of variation calculated from a series of measurements. To assess the overall precision of the method, both system precision and method precision are determined. Jutta Swathi. et al. J Pharm Res, 2025; 14(06): 27-32 © 2012, JPR. All Rights Reserved https://jprinfo.com/ 2.5.4. Robustness The analytical method was subjected to controlled modifications to evaluate its impact on system suitability parameters. The flow rate was systematically varied by ±0.2 mL/min. Additionally, the mobile phase composition was adjusted to two different ratios: 65:35 and 55:45. For each modification, USP tailing factor and USP plate count were measured following six replicate injections of standard solutions. Subsequently, the system suitability parameters were assessed to determine if they remained within acceptable limits 2.5.5. Specificity: The specificity of the method was evaluated by assessing potential interference from placebo on the analyte peak. A placebo sample was injected into the HPLC system following the established test procedure. The resulting chromatogram of the placebo injection was examined to ensure the absence of any peaks at the retention time corresponding to the analyte peak. 3. RESULTS AND DISCUSSION 3.1 Assay of formulation: The assay of the formulation was carried out following the standard procedure in triplicate to ensure accuracy. The amount of each drug (tipiracil and trifluridine) present in the formulation was then calculated using a pre-constructed standard calibration curve. These calculations yielded assay percentages of 101.25% and 101.1% for tipiracil and trifluridine, respectively. Representative chromatograms for the standard solution, the test sample, and a blank were included in the figures. Additionally, the corresponding peak areas for each chromatogram were tabulated in Table 1.Representative chromatograms for standard, test and blank was given in figures 3,4&5. Peak areas were given in table no. 1. 3.2 System suitability System suitability parameters were determined according to ICH guidelines. Plate count was more than 2000, tailing factor was less than 2 and resolution was more than 2. All the system suitable parameters were passed and were within the limits. The results showing system suiability parameters were given in table no. 2 3.3 Validation 3.3.1. Linearity Linearity was assessed using six concentration levels for Trifluridine (ranging from 50 µg/mL to 300 µg/mL) and six concentration levels for Tipiracil (ranging from 22.5 µg/mL to 135 µg/mL). Peak areas were plotted against concentration for each drug, and a calibration curve was constructed. This calibration curve is presented in Figure 3. The correlation coefficient (r²) for both Trifluridine and Tipiracil exceeded 0.99 within the tested concentration ranges. The linearity data is summarized in Table 3. 3.3.2. Accuracy To assess the method's accuracy, the standard addition method was employed at three different concentration levels. Triplicate injections were performed at each level, and the percentage recoveries were subsequently calculated. The mean percent recoveries obtained were 100.01% and 99.55% for Tipiracil and Trifluridine, respectively. A summary of the accuracy data is presented in Table 4 3.3.3. Precision: The method's precision was evaluated by examining both system precision and method precision.Six replicate injections of the same homogeneous standard solution were analyzedforSystem Precision. Peak areas for each injection were determined, and the average area, standard deviation, and % RSD were calculated for both Tipiracil and Trifluridine. The results, presented in Table 5, revealed %RSD values of 0.15% and 0.20% for the retention time of Tipiracil and Trifluridine, respectively. Additionally, the %RSD values for peak area were 0.20% and 0.05% for Tipiracil and Trifluridine, respectively. Six replicate injections of the test solution were analyzedfor Method Precision. Similar to the system precision assessment, peak areas were determined for each injection, and the average area, standard deviation, and % RSD were calculated for both drugs. The results, presented in Table 6, indicated a %RSD value of 0.02% for both the retention time and peak area of both Tipiracil and Trifluridine. 3.3.4. Robustness: The method's robustness was assessed by deliberately introducing variations in flow rate, column oven temperature, and mobile phase ratio. Following each modification, the system suitability parameters were evaluated by injecting standard solutions six times and recording the resulting chromatograms. These parameters were found to be relatively unaffected by the changes, with all values remaining within acceptable limits. Additionally, the percent RSD (% RSD) remained within the established threshold. A summary of the robustness testing results is provided in Table 7. 3.3.5. Specificity A comparison of the standard and sample chromatograms revealed nearly identical profiles with matching retention times for the analytes. The absence of any peaks at the retention time of the analytes in the placebo and sample chromatograms further confirmed the method's specificity. This indicates that there is no interference from either the placebo or the sample matrix at the point of detection for the analytes. Jutta Swathi. et al. © 2012, JPR. All Rights Reserved Table 1: Results of Assay S. No TIPIRACIL Standard Area Sample Area Standard Area 1 1093054 1090989 2818065 2 1092896 1091034 2817988 Mean 1092975 1091012 2818027 Regression equation y = 12700x + 5639.9 y = 14883x % Assay 101.1 % Table 2: Systemsuitability parameters forTrifluridine and Tipiracil SAMPLE Rt Peak Area USP plate count Tipiracil 2.135 1159123 4574 Trifluridine 4.154 2944187 4162 Table 3: Linearity data for Tipiracil and Trifluridine % Level Tipiracil Concentration (μg/ ml) Peak Area Concentration (μ g/ ml) 25% 22.5 284422 50 50% 45 576734 100 75% 67.5 869635 150 100% 90 1158026 200 125% 112.5 1432105 250 150% 135 1713823 300 Table 4: Accuracy data of Trifluridine and Tipiracil % Level Trifluridine Amount Spiked (μg/mL ) Amount recovere d (μg/mL) % Recover y Amount Spiked (μg/mL ) 50% 100 101.02 101.02 45 100 100.05 100.05 45 100 100.15 100.15 45 100 % 200 200.02 100.01 90 200 200.26 100.13 90 200 198.62 99.31 90 150 % 300 298.54 99.5133 135 300 299.47 99.8233 135 300 300.41 100.137 135 Table 5: Method Precision data of Trifluridine and Tipiracil Injection no. Tipiracil Average Peak Area 1089920.667 % RSD 0.02 J Pharm Res, 2025; 14(06): 27 https://jprinfo.com/ Table 1: Results of Assay TRIFLURIDINE Standard Area Sample Area 2818065 2816897 2817988 2817039 2818027 2816968 y = 14883x - 35183 101.25 Table 2: Systemsuitability parameters forTrifluridine and USP plate count USP Tailing 4574 1.21 4162 1.16 Table 3: Linearity data for Tipiracil and Trifluridine Trifluridine Concentration g/ ml) Peak Area 50 711503 100 1446573 150 2200347 200 2943219 250 3685676 300 4428409 Table 4: Accuracy data of Trifluridine and Tipiracil Tipiracil Amount recovere d (μg/mL) % Recover y 44.21 98.2444 44.84 99.6444 45.36 100.8 88.68 98.5333 89.14 99.0444 91.02 101.133 133.58 98.9481 134.98 99.9852 134.58 99.6889 of Trifluridine and Tipiracil Trifluridine 2827528 0.02 Table 6: System Precision data of Trifluridine and Tipiracil Injection no. Tipiracil Average Peak Area 1093497.833 % RSD 0.03 Table 7: Robustness data of Trifluridine and Tipiracil Variation Of Flow rate 0.9ML/MIN 1.1ML/MIN Variation In Buffer Concentration Ratio 60 : 40 70 : 30 Fig 1: Representative Chromatogram of working standard solution Fig 2: Representative Chromatogram of working sample solution J Pharm Res, 2025; 14(06): 27 -32 Table 6: System Precision data of Trifluridine and Tipiracil Tipiracil Trifluridine 1093497.833 2817036 0.03 Table 7: Robustness data of Trifluridine and Tipiracil Tipiracil Trifluuridine Rt. Rt. 2.161 4.422 2.137 3.880 2.161 4.422 2.137 3.880 Fig 1: Representative Chromatogram of working standard solution Fig 2: Representative Chromatogram of working sample solution Jutta Swathi. et al. J Pharm Res, 2025; 14(06): 27-32 © 2012, JPR. All Rights Reserved https://jprinfo.com/ Fig 3: Calibration curve of Tipiracil Fig 4: Calibration curve of Trifluridine 4. CONCLUSION The present work describes the successful development and validation of a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of Tipiracil and Trifluridine in both bulk and tablet dosage forms. The method demonstrated satisfactory performance in terms of accuracy, precision, linearity, and reproducibility.The analysis is performed on a Hypersil C18 column (250mm x 4.6mm, 5µ) with a mobile phase composed of 0.1% ortho phosphoric acid in water adjusted to pH 4 with sodium hydroxide (65%) and acetonitrile (35%). This mobile phase effectively separated Tipiracil and Trifluridine, achieving retention times of 2.1 min and 4.2 min, respectively. A comprehensive validation process was conducted, encompassing system suitability, accuracy, precision, linearity, and ruggedness. The system suitability parameters consistently met established criteria, confirming the method's suitability for the intended assay of Tipiracil and Trifluridine. 5. REFERENCES: 1. World Health Organization. Cancer. Available from: https://www.who.int/news-room/fact-sheets/detail/cancer. Accessed July 3, 2024. 2. American Cancer Society. What Is Cancer? Available from: https://www.cancer.org/cancer/cancer-basics/what-iscancer.html. Accessed July 3, 2024. 3. National Cancer Institute. Cancer Stat Facts: Common Cancer Sites. Available from: https://seer.cancer.gov/statfacts/html/common.html. Accessed July 3, 2024. 4. Mayo Clinic. Cancer. Available from: https://www.mayoclinic.org/diseasesconditions/cancer/symptoms-causes/syc-20370588. Accessed July 3, 2024. 5. Cancer Research UK. What Is Cancer? Available from: https://www.cancerresearchuk.org/about-cancer/what-iscancer. 6. American Cancer Society. Colorectal Cancer: Detailed Guide. 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