INTRODUCTION:

Trifluridine (Figure 1) is a fluorinated pyrimidine nucleoside which is structurally related to idoxuridine and it is also known as trifluoro thymidine. It is an anti-viral drug with chemical name 1- [(2R, 4S, 5R)-4-hydroxy-5 (hydroxyl methyl) oxolan-2-yl]-5-(tri Fluoro-methyl) pyrimidine-2,4-dione¹and molecular formula C₁₀H₁₁F₃N₂O₅and especially for the treatment of viral infections of eye². It acts on viral DNA synthesis and forms defective proteins which increases the mutation rate^3-4. Trifluridine in combination with Tipiracil is available with different brand names in India.

Figure 1: Structure of Trifluridine

Several analytical techniques such as HPLC, LC-MS/MS, microemulsion Electrokinetic chromatography were developed for the simultaneous determination of Trifluridine and Tipiracil in pharmaceutical dosage forms as well as in biological fluids. Valli Kumari et al., developed HPLC method⁵ for the simultaneous determination of Trifluridine and Tipiracil using acetonitrile: 0.1M dipotassium hydrogen phosphate (35: 65) mixture as mobile phase and the linearity was reported as 10-30 µg/ml for Trifluridine and 4.095-12.285 µg/ml for Tipiracil respectively. Hazra et al., developed RP-HPLC method⁶ using Waters HPLC instrument (Model Alliance 2695) with PDA detector and Phenomenex Luna C18 column for the simultaneous determination of Trifluridine and Tipiracil using methanol: water (65: 35) mixture as mobile phase (Detection wavelength 220 nm) and the linearity was reported as 75-375 µg/ml for Trifluridine (Rt 3.2 min) and 15-75 µg/ml for Tipiracil (Rt 5.4 min) respectively. Sahu et al., developed RP-HPLC method⁷ for the simultaneous determination of Tipiracil and Trifluridine using Hypersil C18 column with mobile phase acetonitrile: water: methanol (60: 20: 20) (Detection wavelength 230 nm). The linearity was reported as 66.6-330 µg/ml for Trifluridine (Rt 3.8 min) and 10-50 µg/ml for Tipiracil (Rt 2.8 min) respectively. Mastanamma et al., developed RP-HPLC method⁸ for the simultaneous determination of Tipiracil and Trifluridine using Xterra C18 column with mobile phase consisting of acetonitrile and buffer made up of 0.001% TEA (pH adjusted to 3.0 with ortho phosphoric acid) (60: 40) (Detection wavelength 272 nm). The linearity was reported as 10-50 µg/ml for Trifluridine (Rt 2.016 min) and 4.5-22.5 µg/ml for Tipiracil (Rt 3.471 min) respectively. Kusuma et al., developed RP-HPLC method⁹ for the simultaneous determination of Tipiracil and Trifluridine using Waters Luna C18 column with mobile phase consisting of acetonitrile: ortho phosphoric acid) (50: 50) (Detection wavelength 292 nm). The linearity was reported as 1.02-15.30 µg/ml for Trifluridine (Rt 3.974 min) and 0.41-6.15 µg/ml for Tipiracil (Rt 5.721 min) respectively. Swapna et al., developed RP-HPLC method¹⁰ for the simultaneous determination of Tipiracil and Trifluridine using Kromasil analytical column with mobile phase consisting of potassium dihydrogen phosphate buffer and acetonitrile (30: 70) (pH 2.5) (Detection wavelength 240 nm). The linearity was reported as 50-300 µg/ml for Trifluridine (Rt 2.9 min) and 22.5-135 µg/ml for Tipiracil (Rt 2.3 min) respectively. Prathap et al., developed RP-HPLC method¹¹ for the simultaneous determination of Tipiracil and Trifluridine using Agilent ODS C 18 column with mobile phase consisting of phosphate buffer and methanol (30: 70) (pH 3.0) (Detection wavelength 240 nm). The linearity was reported as 25-125 µg/ml for Trifluridine (Rt 2.262 min) and 15-75 µg/ml for Tipiracil (Rt 4.260 min) respectively.

Asha et al., developed LC-MS/MS/QTOF method¹² for the selective separation of Tipiracil and Trifluridine and their degradants using Symmetry C 18 column with mobile phase consisting of 0.1% TEA and acetonitrile (70: 30) (Detection wavelength 260 nm). The linearity was reported as 3-45 µg/ml for Trifluridine (Rt 2.770 min) and 1.3-18.45 µg/ml for Tipiracil (Rt 5.118 min) respectively. Phani et al., developed RP-HPLC method¹³ for the simultaneous determination of Tipiracil and Trifluridine using Intersil ODS C 18 column with mobile phase consisting of NaClO₄ buffer (pH 4.5) and methanol (15: 85) (Detection wavelength 260 nm). The linearity was reported as 25-125 µg/ml for Trifluridine (Rt 3.4 min) and 15-75 µg/ml for Tipiracil (Rt 7.4 min) respectively. Mohamed et al., developed a new bio analytical microemulsion electrokinetic chromatographic method¹⁴ for the simultaneous determination of Tipiracil and Trifluridine and their metabolites in rat plasma. Trifluridine shows linearity over the concentration 0.2-4.0 µg/ml and 0.1-1.0 µg/ml for Tipiracil respectively (Detection wavelength 205 nm). Spandana et al., developed a spectrophotometric¹⁵ methods in phosphate buffer (pH 7.0, 2.0, 4.0), methanol, borate buffer (pH 9.0), NaOH and water.

Mohammad et al., developed a LC-MS/MS technique¹⁶for the quantification of Trifluridine in human plasma in presence of an internal standard, 𝛽-thymidine. Mobile phase consisting of acetonitrile: methanol: 5 mM ammonium formate (45:40:15) with Phenomenex-RP-C18 column with flow rate 0.8 mL/min are the chromatographic conditions and linearity was shown over the concentration 0.005-2.0 µg/ml. Spandana Yasaswini et al., developed a stability indicating RP-UFLC method¹⁷ for the quantification of Trifluridine using mobile phase mixture, acetonitrile: water (50:50, v/v) with flow rate 0.8 mL/min (UV detection at 261 nm) and the linearity in this method was observed as 1-100 µg/mL.

In the present study a new stability indicating RP-UFLC method has been proposed for the quantification of Trifluridine in ophthalmic preparations and the method was validated as per ICH guidelines.

MATERIALS AND METHODS:

Shimadzu Model UFLC system SPD-M20A 230V with PDA detector and LC- 20AD pumps and C₁₈ Shim-pack GWS HPLC packed column (250 mm × 4.60 mm, 5 μm) was employed for the present study.

10 mM potasium dihydrogen phosphate buffer solution was prepared and the pH was adjusted to 3.5 with dilute tri fluro acetic acid which serves as the aqueous phase. Mixture of acetonitrile and 10 mM potasium dihydrogen phosphate buffer adjusted pH to 3.5 with dilute tri fluro acetic acid (70:30 v/v) was used as mobile phase for the chromatographic elution on isocratic mode with flow rate 1.0 mL/min and the chromatograms were monitored at 272 nm with the help of PDA detector.

25 mg of API Trifluridine was accurately weighed and carefully transferred into a 25 mL volumetric flask and was dissolved in HPLC grade acetonitrile (1000 µg/mL). The resulting solution was sonicated for 30 mins and further dilutions were made with the mobile phase and all the solutions were filtered before use.

Method validation¹⁸:

Linearity, Precision, Accuracy and Robustness:

A series of Trifluridine solutions (0.1-120 µg/mL) were prepared on dilution from the stock solution (1000 µg/mL) with the help of mobile phase, sonicated and each solution was injected three times into the UFLC system and the average peak area from the respective chromatograms was calculated. A calibration graph was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding peak area (n=3) of the chromatogram on the y-axis. The intraday precision studies were conducted on the same day at different equal time intervals and the interday precision studies were conducted on three successive days (Day 1, Day 2 and Day 3) at different concentration levels and the data was statistically analysed. Accuracy studies were performed by spiking the formulation solution with 50, 100 and 150% API solution and thereby the percentage recovery was calculated with the help of calibration curve. Robustness of the method was performed by incorporating small changes in the chromatographic conditions. The percentage relative standard deviation was calculated in all the validation parameters.

Assay of ophthalmic solution (1%):

1% Trifluridine ophthalmic solution is available from Sandoz Falcon Pharmaceuticals (India) and Pfizer Laboratories (India). Two different brands of Trifluridine ophthalmic solution was procured from the pharmacy and Trifluridine was extracted with acetonitrile in a volumetric flask and sonicated for 30 min. The resulting solution was filtered through membrane filter and 20 μL of this formulation solution was injected in to the UFLC system. The peak area of the chromatogram eluted was noted and the percentage purity was determined.

Stress degradation studies¹⁹:

Trifluridine drug solution (50 µg/ml) was exposed to different stress conditions such as acidic hydrolysis, oxidation, alkaline hydrolysis and heat.

Alkaline hydrolysis was performed by exposing Trifluridine solution (50 µg/ml) with 1mL of 0.1N NaOH. This solution was heated for 30 minutes at 80ºC in a thermostat and then neutralized after cooling with 1mL 0.1N HCl solution. The contents were diluted with mobile phase and the resultant solution was injected into UFLC system and the peak area was noted from the recorded chromatogram. Acidic hydrolysis was performed by exposing Trifluridine solution (50 µg/ml) with 1mL of 0.1N HCl. This solution was heated for 30 minutes at 80ºC in a thermostat and then neutralized after cooling with 1mL 0.1N sodium hydroxide solution. The contents were diluted with mobile phase and the resultant solution was injected into UFLC system and the peak area was noted from the recorded chromatogram. During the photolytic degradation Trifluridine solution was exposed to UV light (wavelength 254 nm) for six hours. The resulting solution was diluted with mobile phase (50 µg/ml) and injected into UFLC system and the peak area was noted from the chromatogram. Oxidative degradation was performed by exposing Trifluridine solution (50 µg/ml) with 1mL of 30% H₂O₂. This solution was heated for 30 minutes at 80ºC in a thermostat and cooled. The contents were diluted with mobile phase and the resultant solution was injected into UFLC system and the peak area was noted from the recorded chromatogram. Thermal degradation was performed by heating Trifluridine solution (50 µg/ml) at 80ºC for 30 minutes in a thermostat and cooled. The resulting solution was diluted with mobile phase and injected into UFLC system and the peak area was noted from the recorded chromatogram.

RESULTS AND DISCUSSION:

A new stability indicating RP-UFLC method has been proposed for the quantification of Trifluridine which is used for the treatment of viral infection. Previously reported analytical methods were compared with the present proposed method and some of the parameters were highlighted in Table 1.

Table 1: Literature survey of literature of Trifluridine

Method	Reagent / Mobile phase (v/v)	Linearity (µg/mL)	Ref
Spectrophotometry (Zero order & First derivative methods)	Methanol Water Phosphate buffer (pH 7.0) Phosphate buffer (pH 2.0) Phosphate buffer (pH 4.0) NaOH Borate buffer (pH 9.0)	10-80 10-80 10-80 10-100 10-100 10-100 10-100	15
LC-MS/MS (Human plasma) (Internal standard: 𝛽-thymidine)	Acetonitrile: Methanol: 5 mM Ammonium formate (45:40:15)	0.005-2.0	16
RP-UFLC	Acetonitrile: Water (50:50)	1-100	17
RP-UFLC	Acetonitrile: Potassium dihydrogen phosphate buffer (10 mM) adjusted to pH 3.5 with TFA (70:30)	0.1-120	Present method

Many methods were reported for the combined dosage forms of Trifluridine with Tipiracil. A very few methods were developed for Trifluridine alone. The authors have made different trials with different mobile phases and columns with various flow rates. The chromatograms of placebo, Trifluridine API (10 µg/mL) with the optimised chromatographic conditions was shown in Figure 2. Mobile phase consisting of acetonitrile: 10 mM potassium dihydrogen phosphate buffer adjusted to pH 3.5 with dilute tri fluro acetic acid (70:30 v/v) (Isocratic mode) with 1.0 mL/min flow rate (Detection wavelength 272 nm) are the optimized chromatographic conditions. Trifluridine was eluted at Rt 2.986 min with theoretical plates: 46665.312 and tailing factor: 1.165.

Linearity, Precision, accuracy and robustness:

Trifluridine obeys Beer-Lambert’s law over the concentration range 0.1-120 µg/mL (Table 2) with linear regression equation y = 46195x – 1876.5 (R² = 0.9998) (Figure 3) and the method was validated (% RSD: 0.22-0.68) as per ICH guidelines. The LOQ and LOD values were found to be 0.08934 µg/mL and 0.0257 µg/mL respectively. The method is precise as the % RSD in intraday precision (0.0074-0.1879), interday precision (0.0698-0.1.2272) (Table 3) was found to be less than 2.0%. In the accuracy study the % RSD was found to be 0.762-0.879 (<2) (Table 4) and that of robustness study was 0.0051-0.0993 (Table 5) (<2.0) indicating that the method is accurate and robust.

Table 2: Linearity

Conc. (µg/mL)	*Mean peak area	% RSD
0	0	0
0.1	4851	0.42
0.2	9427	0.26
0.5	24719	0.35
1	47421	0.39
2	93986	0.41
5	237264	0.52
10	473923	0.68
20	941057	0.54
40	1832537	0.38
60	2705841	0.22
80	3661847	0.35
100	4612543	0.43
120	5598721	0.49

*Mean of three replicates

Figure 3: Calibration curve of Trifluridine

Table 3: Precision study

Intraday precision study
Conc. (µg/mL)	Mean peak area	Statistical analysis *Mean peak area ± SD (% RSD)
5	237264	237704.3337 ± 446.6278 (0.1879)
5	238157
5	237692
10	473923	474021.6667 ± 104.4095 (0.0220)
10	474011
10	474131
20	941057	941116.0000 ± 69.7639 (0.0074)
20	941193
20	941098
Interday precision study
Conc. (µg/mL)	Day 1	Day 2	Day 3	Statistical analysis *Mean peak area ± SD (% RSD)
5	237264	238453	238196	237971 ± 625.6189 (0.2629)
10	473923	485412	481968	480434.33 ± 5896.0479 (1.2272)
20	941057	939876	940962	940631.6667 ± 656.1481 (0.0698)

*Mean of three replicates

Table 4: Accuracy study

Spiked conc. (µg/mL)	Formulation (µg/mL)	Total Conc. (µg/mL)	Conc. obtained (μg/mL) ± SD (%RSD)*	% Recovery
10 (50 %)	20	30	29.81 ± 0.2272 (0.762)	99.37
20 (100 %)	20	40	39.74 ± 0.3231 (0.813)	99.35
30 (150 %)	20	50	49.69 ± 0.4368 (0.879)	99.38

*Mean of three replicates

Assay of Trifluridine ophthalmic solution:

Trifluridine 1% ophthalmic solution was procured from two different manufacturers and the proposed RP-HPLC method was applied with the optimized chromatographic conditions. The percentage of purity of Trifluridine was found to be 99.74-99.81. Trifluridine was eluted at Rt 2.988 min with theoretical plates: 4767.860 and tailing factor: 1.155 and the representative chromatogram was shown in Figure 2.

Table 5: Robustness study (40 µg/mL)

Parameter	Condition	*Mean peak area	Mean peak area ± SD (RSD)*
Flow rate (± 0.1mL/min)	1.1	1834421	1832313.667 ± 1818.6752 (0.0993)
	1.0	1832537
	0.9	1829983
Detection wavelength (± 2 nm)	270	1832469	1832565.667 ± 92.8703 (0.0051)
	272	1832537
	274	1832691
Mobile phase composition Acetonitrile: Phosphate buffer (pH adjusted to 3.5 with TFA) (± 5 %)	65: 35	1832995	1832738.667 ± 190.9316 (0.0104)
	70: 30	1832537
	75: 25	1832684

*Mean of three replicates

Stress degradation studies:

Trifluridine (50 µg/mL) was exposed to different stress conditions under the optimized chromatographic conditions. Trifluridine was eluted at 2.991 min with theoretical plates 5649.826 and tailing factor 1.316. During the alkaline hydrolysis Trifluridine was eluted at Rt 2.969 min with theoretical plates 4766.015 and tailing factor 1.222 and about 23.24% of the drug decomposition was observed. During the acidic hydrolysis Trifluridine was eluted at Rt 2.993 min with theoretical plates 5304.517 and tailing factor 1.342 and about 11.11% of the drug decomposition was observed. During the photolyic degradation Trifluridine was eluted at Rt 2.974 min with theoretical plates 4647.827 and tailing factor 1.180 and about 13.91% of the drug decomposition was observed.

During oxidative degradation Trifluridine was eluted at Rt 2.998 min with theoretical plates 4984.514 and tailing factor 1.121 and about 39.87% of the drug decomposition was observed. An extra peak at 2.751 min was also observed during oxidative degradation and the resolution was 2.109. During the thermal degradation Trifluridine was eluted at Rt 2.974 min with theoretical plates 4810.471 and tailing factor 1.152 and about 8.01% of the drug decomposition was observed and in all degradation studies the system suitability parameters were within the acceptable criteria (Table 6) and the corresponding chromatograms obtained during the stress degradation studies were shown in Figure 4.

Table 6: Stress degradation studies of Trifluridine

Condition	R_t (min)	Mean peak area*	% Recovery*	% Drug degradation	Theoretical Plates (>2000)	Tailing factor (<1.5)
Standard drug	2.991	2305841	100	-	5649.826	1.316
Alkaline hydrolysis 0.1N NaOH/80°C/ 30min	2.969	1770003	76.76	23.24	4766.015	1.222
Acidic hydrolysis 0.1N HCl/80°C/30 min	2.993	2049736	88.89	11.11	5304.517	1.342
Photolytic degradation 80°C/30 min	2.974	1987352	86.19	13.91	4647.827	1.180
Oxidative degradation H₂O₂/ 80ºC / 30 min	2.998 2.751	1386469	60.13	39.87	4984.514	1.121
Thermal degradation 80°C/30 min	2.974	2121173	91.99	8.01	4810.471	1.152

*Mean of three replicates


Trifluridine (API)	Alkaline hydrolysis

Acidic hydrolysis	Photolytic degradation

Oxidative degradation	Thermal degradation
Figure 4: Typical chromatograms of Trifluridine (50 µg/mL) during the stress degradation studies

CONCLUSION:

The new validated stability indicating RP-UFLC method developed is simple, precise, accurate and robust. This method is quite simple for the routine analysis of Trifluridine in ophthalmic preparations in quality control department of pharmaceutical industries. The method is quite specific and selective and there is no interference of excipients during the study.

ACKNOWLEDGEMENT:

The authors are grateful to Pfizer Laboratories (India) for providing the gift samples of Trifluridine and there is no conflict of interest.

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Received on 14.04.2022 Modified on 19.05.2022

Research J. Pharm. and Tech. 2022; 15(6):2681-2687.

DOI: 10.52711/0974-360X.2022.00448


Placebo

Typical chromatogram of Trifluridine API (10 µg/mL) (Rt 2.986 min)	Typical chromatogram of Trifluridine (20 µg/mL) ophthalmic solution (Rt 2.988 min)
Figure 2: Typical chromatograms of Trifluridine