New stability indicating ultrafast liquid chromatographic method for the determination of Tilorone in tablets

Gujju Hima Bindu*, Mukthinuthalapati Mathrusri Annapurna, Duvvi Sri Valli

Department of Pharmaceutical Analysis & Quality Assurance, GITAM Institute of Pharmacy, GITAM University, Visakhapatnam-530045, India

*Corresponding Author E-mail: bindureddy.frnd@gmail.com

ABSTRACT:

A new stability indicating RP-UFLC method has been proposed for the determination of Tilorone in bulk and its tablet dosage forms.Chromatographic separation was achieved through C18 Agilent column(150 mm × 4.6 mm i.d., 3.5 µm particle size)using acetonitrile: 0.1 % triethylamine (pH adjusted to 3.2 with orthophosphoric acid)mixture asmobile phase. The UFLC system was monitored at 270 nm on isocratic mode withflow rate 0.5 mL/min and the total run time is 10mins. The method was validated and forced degradation studies were performed. Tilorone has obeyed Beer-Lambert’s law0.1-20 µg/mL with correlation coefficient 0.9999. The LOD and LOQare found to be 0.0104and 0.0316 µg/mL respectively.Tiloronewas subjected to stress degradationstudies. The UFLC method is accurate, precise, sensitive, and reproducible forthe estimation of Tiloronein tablets. The method was validated as per ICH guidelines and found to be specific as the degradants were well separated without interfering with the drug peak.

KEYWORDS: Tilorone, Stability Indicating, UFLC, ICH guidelines

INTRODUCTION:

Tiloroneis a new class of anti-viral drug approved by FDA. It is an orally active interferon inducer. Tilorone is chemically 2, 7-bis-[2(dimethylamino)-ethoxy] - fluorene-9-on.The molecular formula is C₂₄H₃₄Cl₂N₂O₃.2HCl with molecular weight is 483.47. Tilorone(Figure 1) is orange color powder and it is   freely soluble in methanol, water, sparingly soluble in ethanol,dimethyl sulphoxide and dimethyl formamide. Tilorone induced functional changes and Tilorone induced mucopolysaccharidosis were studied in rats^4-5. Analytical methods such as LC-MS/MS⁶and spectrophotometric⁷ methods were reported and no stability indicating liquid chromatographic method has been so far published in the literature and therefore the authors have selected Tilorone and developed UFLC method on isocratic mode and validated as per ICH guidelines⁸. Forced degradation studies⁹ were performed to study the specificity of the method developed for Tilorone.

Figure 1: Chemical structure of Tilorone(TIL)

MATERIALS AND METHODS:

Chemicals and reagents

Tilorone was procured from HONOURS Labs Ltd. (India).Tilorone is available as film coated tablets with brand names AMIXIN and LAVOMAX (Label claim: 60 mg).All chemicals (Acetonitrile, Triethylamine, ortho phosphoric acid)are of AR grade and all solvents are of HPLC grade.The analysis of Tilorone was performed using Shimadzu Model UFLC system(Shimadzu Co., Kyoto, Japan) equipped with SPD M20A prominence photodiode array detector.

Preparation of drug stock and working standard solutions

Accurately 25 mg of Tilorone was taken in a 25mL volumetric flask and volume is made upto the mark with HPLC grade acetonitrile (1000 μg/mL), diluted with mobile phase and filtered through membrane filter.

Method validation

The method was validated by evaluating linearity, recovery, precision, accuracy, system suitability, solution stability, limit of detection (LOD), Limit of quantification (LOQ) and robustness as per ICH guidelines for the determination of Tilorone.

Linearity, precision, accuracy and robustness studies

Different solutions (0.1 to 20 µg/mL) were preparedfrom thestock with mobile phase and 20 µL of each of the solution was injected in to the UFLC system and the peak area of the chromatogram was noted.A graph was plotted using concentration on the x-axis and the mean peak area on the y-axis. Intraday and inter-day precision were studied using three different concentrations of Tilorone on the same day and on three consecutive days respectively. The accuracy of the method was proved by the standard addition method and the recovery values were determined.The robustness study was performed by incorporating small changes in method parameters such as flow rate (± 0.1mL), mobile phase composition (± 2%), pH (± 0.2 units) and detection wave length (275 nm & 265 nm).

Assay of commercial formulation (Tablets)

Two different brands of the tablets were procured andpowdered.Powder equivalent to 25 mg Tiloronewas extracted using the mobile phase.The solution was sonicated for half an hour and filtered through 0.45 mm membrane filter. 20 µL of this solution was injected in to the UFLC system and the peak area was noted from the respective chromatogram.

Stress degradation studies

Stress degradation studies wereperformed to determine the ability of the drug to withstand its properties in the applied stress conditions. Tiloronewas exposed to different stress conditions such as acidic hydrolysis, basic hydrolysis, oxidation and thermaltreatment. Degradation studies were performed by heating the solutions on a water bath for 30 min at 80°. In case of acidic degradation,the drug solution was treated with0.1N HCl, heated, cooled, neutralized with   0.1N sodium hydroxide solution and the solution was made up to volume to the required concentration with the mobile phase. Similarly, the alkaline degradation was performed by treating the drug solution  with 0.1 N NaOH, heated, neutralized with hydrochloric acid and diluted with mobile phase. Oxidative degradation was performed by treating the drug solution with 30% v/v H₂O₂, heated,cooled and diluted with mobile phase. Thermal degradation was performed by heating  the solution  on water bath for 30 min at 80°.  The resultant solution was made to the required concentration with the mobile phase.  All the solutions were filtered through Whatman membrane filter No. 45 and 20 µL of each solution was injected in to the UFLC system and the peak area was noted from the corresponding chromatogram.

RESULTS AND DISCUSSION:

A simple stability indicating reverse phase ultrafast liquid chromatographic method has been developed for the determination of Tilorone in API (active pharmaceutical ingredient) and its tablet dosage forms using C18 agilent column and a mixture of acetonitrile and 0.1%Triethylamine as mobile phase.

Method development and optimization

Agilent C₁₈ column (150 mm × 4.6 mm i.d., 3.5 µm particle size) was selected for the chromatographic study. Chromatography work was performed on isocratic mode using mixture of 0.1 % triethylamine (TEA) and acetonitrile (40: 60, v/v) as mobile phase with flow rate 0.5 mL/min (UV detection at 223nm). The overall run time was 10 min and the study was observed at ambient temperature (25°C ± 2°C).Initially the UFLC system was optimized using C8 Phenomenex column with mobile phase consisting of 0.1 % formic acid: acetonitrile (70: 30, v/v) with flow rate 0.8 and 0.6 ml/min but tailing factor was greater than 2 (Figure 2A and Figure 2B). The Luna phenyl hexane column with Potassium dihydrogen phosphate: ACN mobile phase composition with 1.0 ml/min flow rate resulted a broad peak was (Figure 2C and Figure 2D).Then C 18 Agilent column was tried with ACN: 0.1% trimethylamine (45: 55) with flow rate 0.5 ml/min where the theoretical plates were less than 2000 (Figure 2E) and therefore a slight change in the mobile phase composition (40: 60)finally brought a good symmetrical peak shape with the acceptable system suitability parameters(Figure 2F). The details of the system suitability parameters during the method optimisation and were shown in Table 1.The optimized conditions were shown in Table 2.

Table. 1.Method optimizationof Tilorone (Trial runs)

Column	Mobile phase (v/v)	Flow rate (mL/min)	Rt (min)	Comments	Figure
C8 Phenomenex	0.1 % Formic acid: ACN (70:30)	0.8	2.422	Tailing factor >2	2A
C8 Phenomenex	0.1 % Formic acid: ACN (70:30)	0.6	3.173	Tailing factor >2	2B
Luna phenyl hexane	Potassium dihydrogen phosphate buffer: ACN (46:54)	1	2.886	Broad peak	2C
Luna phenyl hexane	Potassium dihydrogen phosphate: ACN (37:63)	1	2.589	Broad peak Tailing factor>2	2D
C18 Agilent	ACN: 0.1% trimethylamine (45:55)	0.5	2.421	Theoretical plates <2000	2E
C18 Agilent	ACN: 0.1% trimethylamine (40:60)	0.5	2.424	Method optimized	2F

 Table. 2. Optimized conditions for determination of Tilorone

Parameter	Optimized chromatographic conditions
Mobile Phase	0.1 %  Triethylamine and Acetonitrile (40: 60, v/v)
Stationary Phase	C18 Agilent column (150 mm × 4.6 mm i.d., 3.5 µm particle size)
Flow Rate	0.5 mL/min
Detection wavelength	270 nm
Column temp.	(25°±2°C)
Injection Volume	20 µL
Detector	SPD M20A prominence photodiode array detector
Elution	Isocratic mode
Total Run Time Retention time	10 mins 2.440 ± 0.02 mins

Figure 2:Chromatograms obtained during the method optimization of Tilorone (Trial runs)

Method validation

The proposed method was validated by linearity, precision, accuracy, robustness as per the ICH guidelines for the determination of Tilorone. The characteristic chromatograms obtained for the blank and that of Tilorone were shown in Figure 3A and Figure 3B respectively. A calibration curve was drawn taking concentration of Tilorone on the x-axis and the corresponding mean peak area value on the y-axis. Tilorone obeys Beer- Lamberts law over the concentration range 0.1 - 20 µg/mL (Table 3) with linear regression equation y = 44812x + 10588 (correlation coefficient 0.9999 (Figure 4). The LOD and LOQ are found to be 0.0104 µg/mL and 0.0316 µg/mL respectively.

Figure 3: Typical chromatograms a) Blank b) Tilorone standard (10 µg/mL) c) Tilorone tablets (10 µg/mL)

Table. 3. Linearity of Tilorone

Conc.(µg/mL)	*Mean peak area	%RSD
0.1	44215	0.15
0.5	221763.7	0.31
1	448196	0.15
5	2239719	0.25
8	3676269	0.51
10	4472610	0.16
12	5374233	0.31
15	6725076	0.12
20	8936194	0.11

*Mean of three replicates

Figure 4: Calibration curve of Tilorone

Intraday and inter-day precision were studied using three different concentrations of Tilorone on the same day and on three consecutive days respectively. The % RSD was found to be 0.11-0.25 and 0.12-0.38 respectively (<2.0 %) demonstrating that the method is precise (Table 4 & Table 5). The accuracy of the method was proved by the standard addition method and the recovery values were determined. The % recovery of Tilorone and its results of the method are reported in (Table 6). The % RSD was found to be 0.11-0.31 (<2.0 %) with a recovery of 99.19-100.0%. The percentage RSD was found to be 0.07-0.8 (<2.0 %) in robustness study. The system suitability and solution stability were evaluated and the % RSD was less than 2%.

Table. 4.Intraday precision study of Tilorone

Conc. (µg/mL)	*Mean peak area	Statistical Analysis
		*Mean ± SD (% RSD)
5	2244855	2239719 ± 5660.46(0.25)
5	2233650
5	2240652
10	4472610	4467071 ± 7147.90(0.16)
10	4469600
10	4459002
20	8945220	8936194 ± 10176.43(0.11)
20	8925165
20	8938197

*Mean of three replicates

Table. 5.Interday precision study of Tilorone

Conc. (µg/mL)	*Mean peak area			*Mean ± SD (% RSD)
	Day 1	Day 2	Day 3
5	2242562	2239249	2246525	2242779± 3642.83(0.16)
10	4442891	4456577	4477397	4458955 ± 1735.48(0.38)
20	8942126	8921205	8926521	8929951 ± 10874.01(0.12)

*Mean of three replicates

Table. 6. Accuracy study of Tilorone

Conc (µg/mL)			*Mean Conc. (µg/mL) ± SD (%RSD)	% Recovery
Formu Lation	Pure drug	Total
6	3	9	8.91± 0.01 (0.17)	99.50
6	3	9		99.31
6	3	9		99.19
6	6	12	11.96± 0.03 (0.31)	99.99
6	6	12		99.84
6	6	12		99.39
6	9	15	14.98±0.01 (0.11)	100.0
6	9	15		99.83
6	9	15		99.80

*Mean of three replicates

Assay of Tilorone tablets

The proposed RP-UFLC technique was applied for the assay of Tilorone tablets(Label claim: 60 mg)and the percentage of recovery was found to be 99.68- 99.73 (Table 7). The overlay chromatogram obtained for the marketed formulations were shown in Figure 3C.

Table. 7. Assay of Tilorone tablets

Formulation	Label claim (mg)	*Amount found (mg)	*Recovery (%)
Brand I	60	59.84	99.73
Brand II	60	59.81	99.68

*Mean of three replicates

Stress degradation studies

Tilorone was exposed to various stress conditions like acidic, oxidative, thermal and alkaline hydrolysis. Duringoxidation,Tilorone was eluted at 2.388min. About 58.96%of degradation was observed Most of the drug has been degraded in oxidation hydrolysis (77.60%).No degradation peaks have been reported in othe degradations.The percentage of drug degraded in remaining stress condition is less than 10%.The drug peaks were eluted at 2.502 (acid), 2.549 (alkali), 2.458 (thermal) respectively It is confirmed that the drug is sensitive towards oxidation. In all the degradation studies it was found that the drug peak was well separated among the degradants indicating that the method is selective and specific. The system suitability parameters were well in the acceptance criteria (Table 8). The typical chromatograms obtained during degradation studies were shown in Figure 5 and that of 3D chromatograms in Figure 6.

Figure 5:Typical chromatograms of A) Blank B) Tilorone Standard (10 µg/mL) C) Acidic hydrolysis D) Alkaline hydrolysis E) Oxidation F) Thermal degradation

Table. 8. Stress degradation studies of Tilorone

Stress condition Medium /temp /duration	Rt (min)	% Recovery	% Drug degradation	Theoretical plates	Tailing factor
Standard drug	2.430	100	------	3859.214	1.412
Acidic hydrolysis 0.1N HCl/ 80°C/ 30 min	2.502	92.63	7.36	3425.977	1.432
Alkaline hydrolysis 0.1N NaOH/ 80°C/ 30min	2.549	90.98	9.01	3017.447	1.422
Oxidation 30%H2O2/ 80°C/ 30min	2.338	41.03	58.96	3215.507	1.589
Thermal degradation 80°C/30min	2.458	97.50	2.49	2151.273	1.506

CONCLUSION:

The validated stability indicting method developed for the determination of Tilorone is specific and selective and more economical. Tilorone is known to be more sensitive towards basic environment. This method can be excellently applied for the determination of Tilorone in tablets.

ACKNOWLEDGEMENT:

The authors are grateful to HONOURS Labs Ltd. (India) for providing the gift samples of Tilorone. The authors declare that there is no conflict of interest.

Figure 6: 3D chromatograms of Tilorone A) Acidic hydrolysis B) Alkali hydrolysis C) Oxidation D) Thermal degradation

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Received on 06.09.2018           Modified on 15.09.2018

Accepted on 30.09.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3950-3956.

DOI: 10.5958/0974-360X.2018.00726.6