INTRODUCTION:

Trifluridine is an Anti-viral drug. Trifluridine is also known as trifluorothymidine It is a white crystalline powder with molecular formula C₁₀H₁₁F₃N₂O₅and molar mass 296.21 g/mol. It is chemically known as 1-[(2R,4S,5R)-4-hydroxy-5 (hydroxyl methyl) oxolan-2-yl]-5-(tri Fluoro-methyl) pyrimidine-2,4-dione¹. Trifluridine is chemically fluorinated pyrimidine nucleoside that that is structurally related to idoxuridine. Trifluridine eyedrops are used primarily on eye for the treatment of keratitis and keratoconjunctivitis which is caused by herpes simplex virus type 1 and 2. It is also used for treatment and prevention of vaccinia viral infections on eye^2-4. It’s mechanism of action is by inhibition of viral DNA synthesis, it gets incorporated into viral DNA replication that forms defective proteins and causes increased mutation rate^5-7. It was approved for medical use in 1980⁸. In the present study the authors have developed a simple stability indicating RP-UFLC method for the quantification Trifluridine in ophthalmic drops and the method was validated as per ICH guidelines.

Figure 1: Chemical structure of Trifluridine

MATERIALS AND METHODS:

Chromatographic separation was achieved by Shimadzu Model CBM-20A/20 Alite UFLC system (Shimadzu Co., Kyoto, Japan) equipped with SPD M20A prominence photodiode array detector on C18 Agilent column (250 mm × 4.60 mm i.d. 5μm particle size) which is maintained at room temperature. Trifluridine is available as 1% ophthalmic solution with brand name VIROPTIC (Pfizer Laboratories Div Pfizer Inc.) for the treatment of primary kerato conjunctivitis and recurrent epithelial keratitis occurred due to herpes simplex virus, types 1 and 2. VIROPTIC sterile ophthalmic solution contains 1% Trifluridine in an aqueous solution with acetic acid and sodium acetate (buffers), sodium chloride, and thimerosal 0.001% (added as a preservative). The pH range is 5.5 to 6.0 and osmolality is approximately 283 mOsm. 25 mg of Trifluridine (API) was accurately weighed and transferred in to a 25 ml volumetric flask and dissolved in HPLC grade acetonitrile (1000 µg/mL), sonicated and further dilutions are made with mobile phase. Prior to injection all the solutions were filtered through 0.45 µm membrane filter.

Method validation⁹

Linearity, Precision, Accuracy and Robustness

A series of Trifluridine solutions are prepared from stock solution of concentration ranging from10-100 µg/mL. 20 µL of each of these solutions are injected in to the UFLC system. From the chromatographs obtained the mean peak area of trifluridine were calculated and calibration curve was drawn by taking concentration of Trifluridine solutions on x-axis and its corresponding peak area values on y-axis. Interday and intraday precision studies are performed using three different concentration of Trifluridine on three consecutive days and on the same day respectively and %RSD was calculated. The accuracy was evaluated at three concentration levels (50,100 and 150%) and percentage recoveries were calculated. Standard addition and its recovery experiments were conducted to determine the accuracy of the method and for the quantification of Trifluridine in the product and the percentage recovery was calculated. Robustness is assessed by purposely exposing the drug solution to different analytical conditions changing from the original optimized conditions. %RSD was calculated from the summary of the effects obtained and it has to be less than 2.0% indicating that proposed method was robust.

Assay of Trifluridine ophthalmic solution

Trifluridine ophthalmic solution was taken and the solution equivalent to 25 mg of Trifluridine was extracted in methanol in a 25 mg volumetric flask. The solution was sonicated for half an hour and filtered through 0.45 mm membrane filter. 20μL of this solution was injected in to the UFLC system and from the resultant chromatogram the peak area was noted at its retention time.

Forced degradation studies¹⁰

Trifluridine was exposed to different stress conditions like acidic hydrolysis, alkaline hydrolysis, thermal treatment and oxidative hydrolysis. Forced degradation studies were performed to determine the ability of the drug to withstand its properties in applied stress conditions. Acidic degradation was performed by treating the drug solution with 1mL of 0.1N HCl, heated at 80ºC for 30 minutes on water bath and then cooled. The sample is then neutralized with 1mL 0.1N sodium hydroxide solution. The solution is made up to the volume with mobile phase for required concentration. 20 µL of the solution was injected in to the UFLC system. Alkaline degradation was performed by treating the drug solution with 1mL of 0.1N NaOH, heated at 80ºC for 30 minutes on water bath and then cooled. The sample is then neutralized with 1mL 0.1N HCl. The solution is made up to the volume with mobile phase for required concentration. 20 µL of the solution was injected in to the UFLC system. Thermal degradation was performed by heating the drug solution at 80ºC for 30 minutes on a water bath and then cooled. The solution is made up to the volume with mobile phase for required concentration. 20 µL of the solution was injected in to the UFLC system. Oxidative degradation was performed by treating the drug solution with 1 mL of H₂O₂ heated at 80ºC for 30 minutes on a water bath and then cooled. The solution is made up to the volume with mobile phase for required concentration. 20 µL of the solution was injected in to the UFLC system.

RESULTS AND DISCUSSION:

A new reverse phase ultrafast acting stability indicating method was developed for the quantification of trifluridine in ophthalmic drops. A C18 Agilent column (250 mm × 4.60 mm i.d. 5μm particle size) was used. The mobile phase composition is Acetonitrile: water (50:50 v/v) and at flow rate of 0.8 ml/min with UV detection at 261 nm was used for the determination of Trifluridine by which sharp peal was observed at 2.396 min (Run time is 10 min). The optimized chromatographic conditions were shown in Table 2.

Method optimization

During the method optimization C18 Agilent column was tried (Table 1) with Acetonitrile: water (70:30) with flow rate of 1ml/min where the retention time is less than 2 (Figure 2A) and therefore mobile phase composition is changed to Acetonitrile: water (65:35) with flowrate 1ml/min, even then the retention time is less than 2 (Figure 2B). Later mobile phase composition is changed to Acetonitrile: water (60:40) with 1ml/min flowrate in which retention time is less than 2 (Figure 2C) and therefore the mobile phase composition is changed to Acetonitrile: water (40:60) in which the retention time is less than 2 (Figure 2D). Later mobile phase composition is changed to Acetonitrile: water (40:60) with 0.8ml/min flowrate in which theoretical plates are less than 2000 (Figure 2E) and then mobile phase composition is changed to Acetonitrile: water (50:50) with 0.8 ml/min flow rate by which the method was optimized (Figure 2F) (Table 2).

Table 1: Method optimization of Trifluridine (Trial runs)

Flow rate (mL/min)	Mobile phase ratio	R_t(min)	Remarks	Figure
1	Acetonitrile: water (70:30)	1.689	Retention time is less than 2	2A
1	Acetonitrile: water (65:35)	1.714	Retention time is less than 2	2B
1	Acetonitrile: water (60:40)	1.743	Retention time is less than 2	2C
1	Acetonitrile: water (40:60)	1.787	Retention time is less than 2	2D
0.8	Acetonitrile: water (40:60)	2.244	Theoretical plates <2000	2E
0.8	Acetonitrile: water (50:50)	2.396	Method optimized	2F

Figure 2: Typical chromatographs of Method optimization of Trifluridine (Trial runs)

Table 2: Optimized chromatographic conditions

Parameter	Optimized chromatographic conditions
Mobile phase	Acetonitrile: Water (50:50)
Stationary phase	C18 Agilent column
Flow rate	0.8 ml/min
Detection wavelength	261 nm
Column temp.	(25°±2°C)
Injection volume	20 μL
Detector	PDA
Elution	Isocratic mode
Total run time	10 min
Retention time	2.396

Linearity, Precision, accuracy and robustness

Trifluridine has shown the linearity over the concentration range of 10-100 µg/mL (Table 3) with linear regression equation y = 66167x + 13368 (R² = 0.9999). The system suitability parameters for the trifluridine has shown the tailing factor was less than 2 (or <1.5-2.0) and theoretical plates are more than 2000. Interday and intraday precision studies are performed using three different concentration of Trifluridine on three consecutive days and on the same day respectively and %RSD was found to be Interday precision (0.213-0.788), intraday precision (0.164-0.572) (Table 4) respectively (<2.0) demonstrating that the method is precise. The accuracy was evaluated by standard addition method and percentage recoveries were found to be 99.50-99.78% (Table 5). The robustness of the assay method was established by introducing small changes in the chromatographic conditions which include detection wavelength (259 and 263 nm), percentage of acetonitrile in the mobile phase (42% and 48%) and flow rate (± 0.1 ml/min). Robustness of the method was studied using 10 μg/mL of Trifluridine (Table 6) and the % RSD was found to be 0.251-0.396 (<2.0). The percentage of purity of Trifluridine was found to be 99.24-99.74.

Table 3: Linearity of Trifluridine

Conc.(µg/ml)	*Mean peak area
0	0
10	669425
20	1337450
40	2657500
60	4012432
80	5345398
100	6583130

*Mean of three replicates

Table 4: Precision study of Trifluridine

Conc.(µg/ml)	*Mean peak area	*Mean ± SD (% RSD)
Intraday precision
10	669521	669510 ± 110.1514 (0.164)
20	1337439	1337376 ± 7655.935 (0.572)
40	2657492	265724 ± 591.9741 (0.227)
Interday precision
10	669425	669485 ± 5277.61 (0.788)
20	1337450	1338122 ± 7498.53 (0.561)
40	2657500	2658547 ± 5681.112 (0.213)

*Mean of three replicates

Table 5: Accuracy study of Trifluridine

Spiked conc. (µg/ml)	Formulation (µg/ml)	Total conc. (µg/ml)	Conc. obtained (μg/mL) ± SD (%RSD)*	% Recovery
12.5 (50 %)	25	37.5	37.39 ± 0.1320 (0.329)	99.70
25.0 (100 %)	25	50.0	49.89 ± 0.2090 (0.419)	99.78
37.5 (150 %)	25	62.5	62.19 ± 0.1560 (0.251)	99.50

*Mean of three replicates

Table 6: Robustness study of Trifluridine

Parameter	Condition	*Mean peak area	Mean peak area ± SD (RSD)*
Flow rate (± 0.1ml/min)	0.7	677124	671596 ± 48.317 (0.719)
	0.8	669485
	0.9	658179
Detection wavelength (±2 nm)	259	669548	667007 ± 42.9506 (0.643)
	261	669425
	263	662048
Mobile phase Composition Acetonitrile: Water	42: 58	668173	668463 ± 12.541 (0.187)
	50: 50	669425
	48: s52	667167

*Mean of three replicates

Forced degradation studies

Trifluridine was eluted at 2.383 min. Trifluridine has undergone acidic hydrolysis (11.79 %), thermal degradation (28.73%), alkaline hydrolysis (8.97%) and oxidative degradation (53.09). During acidic hydrolysis an extra peak was observed at 3.155 min along with the drug peak at 2.383 min, for thermal hydrolysis an extra peak was observed at 3.050 min along with drug peak at 2.379 min and for oxidative hydrolysis the extra peak was observed at 2.102 min along with drug peak at 2.408 min. Trifluridine was found to be more sensitive towards oxidative and thermal conditions and more resistant towards acidic and alkaline conditions as the degradation was less than 20%. The system suitability parameters were well in the acceptance criteria (Table 7). The individual chromatograms observed during the forced degradation studies were shown in Figure 3.

Table 7: Stress degradation studies of Trifluridine

Stress condition	R_t (min)	% Recovery*	% Drug degradation	Theoretical Plates (>2000)	Tailing factor (<1.5)
Standard drug	2.383	100	-	3972.737	1.352
Acidic degradation 0.1N HCl/80°C/30 min	2.385 3.155	88.21	11.79	3691.665	1.223
Alkaline degradation 0.1N NaOH/ 80°C/ 30 min	2.376	91.03	8.97	3827.982	-
Thermal degradation 80°C/30 min	2.379 3.050	71.27	28.73	2999.546	1.146
Oxidative degradation H₂O₂/ 80ºC / 30 min	2.408 2.102	46.91	53.09	3107.159	1.160

*Mean of three replicates

Figure 3: Typical chromatographs of A) Mobile phase B) Trifluridine (API) (20 µg/ml) C) Viroptic D) Acidic degradation

E) Thermal degradation F) Oxidative degradation G) Alkaline degradation

CONCLUSION:

The RP-UFLC technique so developed for the determination of Trifluridine was validated as per ICH guidelines and found to be simple, economical and robust for the quantification of Trifluridine. Trifluridine was found to be more sensitive towards oxidative and thermal conditions and the method is specific.

ACKNOWLEDGEMENT:

The authors are grateful to M/s GITAM (Deemed to be) University, Visakhapatnam for providing the research facilities and Pfizer Laboratories Div Pfizer Inc. and Biophore pharmaceuticals (India) for providing the gift samples of Trifluridine. The authors declare no conflict of interest.

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Received on 15.03.2020 Modified on 29.04.2020

Research J. Pharm. and Tech 2020; 13(6): 2881-2885.

DOI: 10.5958/0974-360X.2020.00514.4