A Review on RP-HPLC Method for the Simultaneous Estimation of Trifluridine & Tipiracil in Bulk and its Tablet Dosage Form

Abstract

Trifluridine is an antiviral agent primarily used to treat eye infections, particularly herpes keratitis, and is also a component of the combination drug trifluridine/tipiracil, utilized in the treatment of advanced cancers, including metastatic colorectal and gastric cancer. This combination is designed for patients who have not responded to prior therapies. Tipiracil (TAS-102) is an oral chemotherapy drug used to treat previously treated metastatic colorectal cancer (mCRC) and gastric cancer. Reviews highlight that it improves overall and progression-free survival for patients refractory to other therapies, but it has a manageable side effect profile primarily involving hematological (anemia, neutropenia, thrombocytopenia) and gastrointestinal issues. A satisfactory linear response was confirmed across a specified concentration range for both compounds, showcasing a high correlation coefficient (\(r^{2}\) value of 0.999 or higher). The accuracy and precision metrics were promising, with percentage recovery values falling within the acceptable range of 97-102% and relative standard deviation (RSD) values for intra-day and inter-day precision remaining below 2%. This indicates a high level of reliability and reproducibility for the analytical method. The specificity of the method was demonstrated, showing no interference from common excipients or degradation products when stability-indicating studies were conducted. Furthermore, robustness was verified by observing minimal variations in results when minor adjustments to chromatographic conditions were applied. In conclusion, the RP-HPLC method proposed in this study can be confidently utilized for routine quantitative analysis of trifluridine and tipiracil in various pharmaceutical formulations.

Keywords

Introduction

Trifluridine (Figure 1) is an antiviral agent primarily used to treat eye infections, particularly herpes keratitis, and is also a component of the combination drug trifluridine/tipiracil, utilized in the treatment of advanced cancers, including metastatic colorectal and gastric cancer. This combination is designed for patients who have not responded to prior therapies (1).

Figure 1: Trifluridine

Trifluridine works as a nucleoside analog that integrates into the DNA of replicating cells, leading to the inhibition of DNA synthesis and subsequent cellular proliferation. It serves as a powerful alternative for treating herpes simplex virus keratitis, proving especially effective in challenging cases. In terms of oncology, when combined with tipiracil, trifluridine remains active by preventing its breakdown, enhancing its therapeutic efficacy. This combination is indicated for patients with metastatic colorectal or gastric cancer who have undergone at least two previous systemic chemotherapy treatments. Clinical trials substantiate that trifluridine/tipiracil significantly improves overall survival and progression-free survival, making it a crucial option for patients with limited treatment avenues (2, 3). The therapy is associated with a manageable side effect profile, with gastrointestinal symptoms such as nausea, diarrhea, and vomiting, along with hematological issues like neutropenia, leukopenia, and anemia being the most frequently reported adverse events (4). These side effects can often be addressed through dose modifications and supportive care. Notably, myeloid toxicity, particularly neutropenia, frequently acts as a dose-limiting factor in this regimen (5). Additionally, the synergy between trifluridine/tipiracil and bevacizumab offers enhanced benefits for treating metastatic colorectal cancer, contributing to a favorable safety profile while improving therapeutic outcomes (6).

Tipiracil (Figure 2) is an oral chemotherapy agent specifically designed for patients with previously treated metastatic colorectal cancer (mCRC) and gastric cancer. Clinical reviews indicate that this medication improves both overall survival (OS) and progression-free survival (PFS) for individuals who are refractory to other treatment options. The side effect profile of TAS-102 is considered manageable, primarily involving hematological effects such as anemia, neutropenia, and thrombocytopenia, along with gastrointestinal issues like nausea, vomiting, and diarrhea (7, 8).

Figure 2: Tipiracil

The efficacy of TAS-102 has been notable, as it has demonstrated the ability to prolong survival in mCRC and advanced gastric cancer patients whose condition has worsened following other therapies. It serves as a critical treatment choice for heavily pre-treated patients with advanced disease, providing a feasible option for those who have undergone multiple prior treatment regimens. Real-world studies suggest that the effectiveness of TAS-102 aligns closely with results seen in clinical trials such as the RECOURSE trial, although it is noted that some data may indicate slightly reduced outcomes when compared to Asian studies (9, 10). Additionally, the inclusion of TAS-102 does not detrimentally affect health-related quality of life, and it has been associated with postponing the decline in performance status for patients. When used in combination with bevacizumab, TAS-102 has exhibited promising results in treating mCRC, with ongoing research continuing to explore its full potential. In terms of safety, hematological side effects are the most frequently encountered serious adverse effects, particularly grade 3 or higher neutropenia, thrombocytopenia, and anemia. While gastrointestinal side effects are also prevalent, the overall occurrence of grade 3 infections remains low among the treated patient population. Management of these adverse effects is typically effective and can include dose adjustments, temporary treatment interruptions, or the application of supportive care strategies. The drug works through its two primary components: trifluridine and tipiracil. Trifluridine, the active ingredient, integrates into the DNA of cancer cells, thereby disrupting their functions. Tipiracil inhibits the enzyme thymidine phosphorylase, which prevents the disintegration of trifluridine, enhancing its bioavailability and therapeutic efficacy. Due to its short half-life, TAS-102 is administered on a structured schedule, taken twice daily to ensure optimal treatment outcomes (11, 12).

2. REVIEW ON THE METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF TRIFLURIDINE & TIPIRACIL IN BULK AND ITS TABLET DOSAGE FORM

Kallam SD et al., 2025 developed and validated of a stability-indicating UPLC-MS/MS method for the simultaneous determination of trifluridine, tipiracil hydrochloride and their impurities. The chromatographic separation was achieved on a Waters UPLC using the Acquity BEH Phenyl column (100 mm ×2.1 mm, 1.7 µm) with a mobile phase comprising methanol and ammonium formate buffer (50:50, v/v) at a flow rate of 0.5 mL/min. The detection was carried out at the wavelength of 257 nm. The validation of the developed method adhered to ICH guidelines, evaluating parameters such as specificity, linearity, precision, accuracy, robustness and forced degradation. The method exhibited excellent linearity (R2 > 0.999), high precision (%RSD < 1%) and recoveries ranging from 99.54% to 100.90%. The method was demonstrated to be robust against variations in flow rate and mobile phase composition, showing reproducibility across analysts. Forced degradation studies under acidic, alkaline, oxidative, thermal, photolytic and hydrolytic conditions confirmed the method’s stability-indicating capability. Degradation products were identified and characterized using LC–MS/MS, providing enhancing its application for stability testing, which is crucial for ensuring the quality, safety and efficacy of pharmaceutical  formulations. The developed method has been reported to be reliable and suitable for routine analysis of trifluridine, tipiracil hydrochloride and their impurities/related substances in both bulk drug and dosage forms (13).

Balekundri A et al., 2025 optimized method yielded reliable Rf values of 0.64 for TIP and 0.91 for TRI. Validation per ICH Q2 (R1) guidelines confirmed excellent linearity (R² = 0.9944 for TIP and R² = 0.9988 for TRI), low detection limits (0.0011 µg/mL for TIP and 0.0022 µg/mL for TRI), and high precision (intra-day %RSD <0.74, inter-day %RSD <0.92). Robustness testing demonstrated minimal variability in Rf values (%RSD <0.28). Environmental sustainability was assessed using ComplexGAPI, AGREE, and BAGI tools. The developed method achieved an AGREE score of 0.81, an Eco-Scale score of 86, and a BAGI score of 80, highlighting its eco-friendliness and practical applicability. This study demonstrates an efficient, precise, and environmentally sustainable analytical method for TRI and TIP quantification, aligning with green chemistry principles and ensuring minimal environmental impact (14).

Shelke RU et al., 2025 developed and validated a robust, efficient HPLC method for simultaneous estimation of Dolutegravir (DLV) and Rilpivirine (RLV) in bulk and tablet formulations. Optimization using Central Composite Design (CCD) within a Design of Experiments (DoE) framework established critical chromatographic parameters on a Phenomenex C18 column (250 x 4.6 mm, 5 μm) with PDA detection at 272 nm. The optimized mobile phase comprised acetonitrile and phosphate buffer (66:34 % v/v) at pH 3.1, with a flow rate of 0.9 mL/min. The method showed retention times of 2.75 min (DLV) and 3.44 min (RLV), resolution > 2.0, and capacity factors (K′) within standard range, ensuring peak separation. Forced degradation studies demonstrated specificity, with drug degradation ranging from 3.25% (photolytic) to 23.9% (alkali), confirming the method’s stability-indicating capability. The quadratic and interaction models accounted for over 90% of response variability (R² > 0.90), revealing significant effects of pH, flow rate, and organic phase composition. Validation per ICH Q2(R2) guidelines showed accuracy between 98–102%, precision (%RSD) below 2%, and high robustness. This approach bridges gaps in existing literature by integrating comprehensive degradation profiling, statistical optimization, and design space modeling with risk assessment (15).

Zhang R et al., 2024 conducted a study in which rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed and fully validated for the simultaneous determination of TPI, FTD, and the metabolite 5-trifluoromethyluracil (FTY) of FTD in human plasma. The plasma samples were prepared by protein precipitation. The chromatography separation was performed using ACE Excel 3 AQ (100 × 2.1 mm i.d., 1.7 µm, ACE, England) column protected by a security guard cartridge (4.0 × 2.0 mm i.d., 5 µm, Phenomenex, USA) with a gradient elution of 0.05% acetic acid in water and methanol at a flow rate of 0.35 mL/min. The MS/MS analysis was performed by using multiple reaction monitoring with the segmented polarity (positive for TPI: m/z 243.1→183.0, and negative for FTD: m/z 295.1→252.0 and FTY: m/z 178.9→158.9) electrospray ionization mode. The segmented polarity mode was designed to achieve two advantages: better sensitivity and simultaneous determination of the analytes with different ion polarities. The calibration ranges were as follows: 1.00–250 ng/ for TPI, 8.00–8000 ng/mL for FTD and 5.00–1250 ng/mL for FTY. The selectivity, accuracy, precision, matrix effect, recovery, carryover, dilution integrity and stability test results meet ICH acceptance criteria. The method was evaluated using the RGB model and successfully applied to a clinical study in patients with solid tumors. For TPI, FTD and FTY, the maximum plasma concentration was 137–147 ng/mL, 6160–6240 ng/mL and 724–725 ng/mL, respectively; the plasma elimination half-life was 1.69–1.78 h, 1.70 h, and 3.09–3.14 h, respectively, after an oral administration of 60 mg TAS-102 (16).

Salem H et al., 2024 established a quick and practical fluorometric technique for trifluridine analysis. The approach relied on the drug's complex formation with the zinc ion to produce a high-fluorescence product. The fluorescence was further enhanced by adding sodium dodecyl sulfate, and it was observed at 450 nm following excitation at 400 nm. With a determination coefficient of 0.9994, the association between emission intensity and trifluridine concentration was linear between 1 and 125 ng mL−1. The quantitation limit was 0.987 ng mL−1 while 0.333 ng mL−1 was the detection limit. The buffer type, pH and concentration, type of surfactant and concentration, and finally the diluting solvent were among the reaction conditions that were closely examined. With great precision and reliability, the drug in question was quantified using this method in dosage formulations. The proposed method's level of greenness was assessed using two methodologies: the analytical greenness metric (AGREE) and the Green Analytical Procedure Index (17).

Kumar KS et al., 2024 developed and validated a new reversed-phase high performance liquid chromatographic technique. It is straightforward, accurate, fast, selective, and stable. Based on a Phenomenex Gemini C18 (4.6×250mm) 5µ column, the procedure operates. Isocratic elution using a 65:35% v/v methanol:TEA buffer ratio, pumped at 1.0 ml/min, and UV detection at 230 nm is used to produce the separation. The analysis is conducted with the column maintained at 40°C. About six minutes have passed in total. According to the guidelines set out by the International Conference on Harmonisation (ICH), the technique has been validated for specificity, accuracy, precision, and linearity as well as for robustness and ruggedness, system adaptability, limit of detection, and limit of quantitation. For the measurement of trifluridine and tipiracil between 10-50 µg/ml and 20-100 µg/ml respectively, the technique is linear and accurate. Additionally, good outcomes are also demonstrated in terms of robustness, intra-day and inter-day accuracy (&lt;2%), and mean percentage recovery (100.37% for trifluridine and 100.34% for tipiracil). This approach has the advantages of adequate accuracy and fine resolution with sharper peaks. The findings show that the approach is appropriate for regular quality control testing of tablet formulations that are sold (18).

Afreen H et al., 2023 developed and validated a Analytical Method for Trifluridine and Tipiracil in bulk and Combine Dosage Form by RP-HPLC, New method was established for simultaneous estimation of Trifluridine and Tipiracil by RP-HPLC method. The chromatographic conditions were successfully developed for the separation of Trifluridine and Tipiracil by using Symmetry C18 5µm (4.6 x 150mm), flow rate was 1.0 ml/min, mobile phase ratio was Phosphate buffer (0.02M) pH-3.8: Methanol: Acetonitrile (60:20:20%v/v), detection wavelength was 260nm. The retention times of Trifluridine and Tipiracil were found to be 2.324mins and 4.314mins respectively. The % purity of Trifluridine and Tipiracil was found to be 99.865% and 99.658% respectively. The analytical method was validated according to ICH guidelines (ICH, Q2 (R1)). The linearity study n Trifluridine and Tipiracil was found in concentration range of 0µg-36µg and 0µg-39µg and correlation coefficient (r2) was found to be 0.9995 and 0.9998, % recovery was found to be 100.280, %RSD for repeatability was 0.174 and 0.709, % RSD for intermediate precision was 0.093 and 0.937 respectively. The precision study was precise, robust, and repeatable. LOD value was 1.377 and 1.079, and LOQ value was 4.174 and 3.272 respectively. Hence the suggested RPHPLC method can be used for routine analysis of Trifluridine and Tipiracil in API and Pharmaceutical dosage form (19).

El-Gendy M et al., 2023 developed and validated a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of tipiracil (TIP), trifluridine (FTD), and their metabolites, 5-trifluoromethyluracil (FTY) and 5-carboxy-2′-deoxyuridine (5CDU), in rat plasma. This method is highly sensitive, specific, and fast. Paracetamol (PAR) is used as an internal standard (IS). Using acetonitrile-induced protein precipitation, the analytes were extracted from a plasma sample and separated on a Waters BEH C18 (1.7 μm particle size, 50 mm × 2.1 mm ID) column protected by a security guard cartridge (C18, 4 × 2.0 mm). The isocratic mobile phase was made up of methanol and water containing 0.1% formic acid (80:20, v/v) at a flow rate of 0.5 mL/min for 4 min. The quantification was performed using a positive electrospray ionization (ESI) interface and a multiple-reaction monitoring (MRM) mode. The MRM transitions employed were m/z 242.96 → 182.88 for TIP, 296.96 → 116.86 for FTD, 180.98 → 139.85 for FTY, 272.96 → 156.86 for 5CDU, and 151.97 → 92.68 for IS. The validated method complied with the guidelines set by the US-FDA over on a linear concentration range of 5–4000 ng/mL for FTD, FTY, and 5CDU, and 5–1000 ng/mL for TIP. The coefficient of determination (r2) was equal to or greater than 0.997. The corresponding lower limits of detection (LLOD) were 1.5 ng/mL for FTD, FTY, and 5CDU and 1.0 ng/mL for TIP. The recoveries of all analytes from rat plasma ranged from 88.67% to 112.18%, and the mean relative standard deviation (RSD) of accuracy and precision result was less than or equal to 6.84%. FTD, FTY, 5CDU, and TIP demonstrated adequate stability throughout the various circumstances examined. Additionally, no matrix effects were identified for any of the analytes. The assay was effectively utilized to conduct a pharmacokinetic study in rats following the oral administration of FTD and TIP at a dosage of 5.6 mg/kg, with a ratio of 1:0.5 for FTD and TIP, respectively. This indicates that the suggested approach is suitable for future clinical research. The pharmacokinetic parameters Cmax (maximum concentration), Tmax (time to reach maximum concentration), t1/2 (half-life), AUC0-24 (area under the concentration–time curve from 0 to 24 h), AUC total (total area under the concentration–time curve), Ke (elimination rate constant), Vd (volume of distribution), and CL (clearance) of all analytes were assessed. The assay developed exhibits significant advancements compared to earlier bioanalytical methods documented in the literature. These improvements include high sensitivity, specificity, and efficacy in high throughput analysis of complex matrices. Additionally, the assay offers a shorter run time and smaller sample volume (50 μL) (20).

3. FUTURE SCOPE

The future development and validation of RP-HPLC methods for trifluridine and tipiracil will focus on improving efficiency, sensitivity, environmental sustainability, and regulatory compliance. Key areas of advancement include: Emphasis will be placed on devising faster methodologies that incorporate environmentally benign solvents, such as ethanol, and reduce run times to minimize costs and waste. Adopting Ultra-Performance Liquid Chromatography (UPLC) or Ultra-High Performance Liquid Chromatography (UHPLC) is expected to enhance separation efficiency, speed, and sensitivity by utilizing smaller particle size columns. There will be continued efforts to refine quantification methods for biological fluids, such as human plasma and urine, thereby supporting pharmacokinetic and in vivo studies, potentially employing advanced detectors like LC-MS/MS to achieve higher sensitivity and specificity. Future research may concentrate on extensive stress testing and the application of advanced analytical techniques (e.g., LC-MS/MS/QTOF) to more effectively characterize and identify potential degradation products under diverse conditions. The integration of QbD principles and DoE will become increasingly significant for systematic, robust, and optimized method development, ensuring consistent performance across a variety of conditions. Implementing software tools for the automation of method optimization and predictive modeling could significantly streamline the development and validation processes, thereby minimizing manual labor and reducing the likelihood of errors. There will be a concentrated effort on creating highly specific methods capable of separating and quantifying both active pharmaceutical ingredients and their impurities, an essential requirement for meeting regulatory standards. These innovations aim to deliver more robust, cost-effective, and efficient analytical tools for quality control and research pertaining to trifluridine and tipiracil formulations.

4. CONCLUSION

Significantly, the chromatographic separation achieved a good resolution between trifluridine and tipiracil within a short runtime, utilizing an optimized mobile phase and column. A satisfactory linear response was confirmed across a specified concentration range for both compounds, showcasing a high correlation coefficient (\(r^{2}\) value of 0.999 or higher). The accuracy and precision metrics were promising, with percentage recovery values falling within the acceptable range of 97-102% and relative standard deviation (RSD) values for intra-day and inter-day precision remaining below 2%. This indicates a high level of reliability and reproducibility for the analytical method. The specificity of the method was demonstrated, showing no interference from common excipients or degradation products when stability-indicating studies were conducted. Furthermore, robustness was verified by observing minimal variations in results when minor adjustments to chromatographic conditions were applied.  In conclusion, the RP-HPLC method proposed in this study can be confidently utilized for routine quantitative analysis of trifluridine and tipiracil in various pharmaceutical formulations.

5. CONFLICT OF INTEREST

None

REFERENCES

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