Silicosis is a fibrous lung disease caused by the inhalation of crystalline silica dust^1,2. It is a debilitating, progressive, non-reversible and sometimes fatal lung disease. The great damage of silicosis has been gradually recognized and many drugs have been synthesized for the treatment of silicosis.³ Tiloronoxim is a derivative of Tilorone recently synthesized for the treatment of silicosis. The preclinical study demonstrated the efficacy of tiloronoxim with low chronic toxicity.⁴ It is rapidly and widely distributed in the body and is excreted in the urine. It is metabolized in different metabolites in humans and among these Tilorone (Figure 1) is pharmacologically active⁵.

Figure 1: Chemical structure of Tilorone dihydrochloride

Tilorone is a new class of antiviral drugs, approved by the FDA. It is an orally active inducer of interferon.⁶ Specifically induces a prolonged and unusual response to prolonged interferon that is uncommon compared to other synthetic inducers.^7-10 Tilorone is an orange powder that is freely soluble in methanol, water and moderately soluble in ethanol, dimethylsulfoxide, and dimethylformamide. Chemically, it is 2,7-bis- [2 (dimethylamino) -ethoxy] -fluorene-9-one with molecular formula C₂₅H₃₄N₂O₃ (molecular weight: 410.55g/mol).

Few articles have been published for the determination of tiloronoxim or Tilorone in biofluids. The determination of Tilorone in human urine has been reported by HPLC-MS/MS¹¹ and a spectrophotometric derived method.¹² No document has been reported to quantify impurities related to the tilorone process to date. This document describes a sensitive, specific and rapid HPLC method for the determination of Tilorone impurities and the methods have been validated according to the ICH guidelines.¹³

EXPERIMENTAL:

Chemicals and solvents:

The pure drug Tilorone was obtained as a gift sample from MSN Laboratories, Hyderabad, India. Tilorone tablets were purchased at the local pharmacy. Methanol, acetonitrile and HPLC grade water were purchased from SD Fine Chem, Mumbai, India. All other chemicals used were of AR grade.

HPLC–PDA instrumentation and chromatographic conditions:

The HPLC system was an LC Waters (Waters, Milford, MA, USA) composed of a quaternary gradient system (600 controllers), in-line degasser (Waters, model AF), photodiode array detector (water, model 2998) and automatic sampler (Waters, model 717 plus). The data was processed with the Empower Pro software (Waters, Milford, MA, USA). The chromatographic separation test was performed with a Zorbax SB-phenyl analytical column (internal diameter 150mm × 4.6mm, particle size 5μm) maintained at 40°C. mobile phase A: 20mM potassium dihydro phosphate + 2ml of triethylamine, pH 2.30 and mobile phase B: acetonitrile, methanol and water 60: 20: 20% v/v. A gradient program was followed with a run time of 55 minutes at a flow rate of 1.0 ml/min. The detection wavelength was 269nm. 20mM potassium dihydro phosphate buffer and methanol in the ratio of 70:30 %v/v were used as diluent.

Preparation of Mobile phase:

Mobile phase A:

4.76g of potassium dihydrogen phosphate was carefully weighed and dissolved in 500ml of distilled water, sonicated for 10 minutes to dissolve and the volume was made up to 1000ml with distilled water. 2ml of triethylamine were added, the pH of the solution was adjusted to 2.30±0.05 with a solution of diluted orthophosphoric acid. The solution was filtered through a 0.22µm membrane filter.

Mobile phase B:

A degassed mixture of acetonitrile, methanol and water 60: 20: 20% v/v was prepared.

Diluent:

A mixture of 20mM potassium dihydro phosphate buffer and methanol in a ratio of 70: 30% v/v is used as a diluent in the preparation of analysis solutions.

Preparation of stock solution and analysis solutions:

A stock solution (1000mg/ml) was prepared by transferring the appropriate amount of bulk Tilorone drug to a 100ml volumetric flask containing about 25ml of diluents and the solution was sonicated for about 10 minutes or until the solid it has completely dissolved. Then the volumetric flask was filled to mark with diluent. The Tilorone stock solution was diluted in series with diluent to provide working solutions of the desired concentrations. A stock solution of impurities (TLHC01 and TLHC02) at 1mg/ml in diluents was also prepared; all solutions were stored at room temperature.

Stress studies/specificity:

Specificity is the ability to measure the response in the presence of its possible impurities. The forced degradation of the pharmacological substance of Tilorone was carried out under conditions of hydrolytic and acid/base oxidative stress.

Acid degradation:

API, placebo and placebo spiked with Tilorone were subjected to reflux with 15ml of 3M hydrochloric acid for 15 minutes at 80°C. Samples subjected to stress (pH 1.30-1.50) were cooled, neutralized with 3 M sodium hydroxide and diluted with a final concentration of 0.95 mg/ml in case of API and placebo spiked with Tilorone.

Alkaline degradation:

API, placebo and placebo spiked with Tilorone were subjected to reflux with 15ml of 3M sodium hydroxide for 15 minutes at 80°C. Samples subjected to stress (pH 11.00 to 12.50) were cooled, neutralized with 3 M hydrochloric acid and diluted with a final concentration of 0.95mg/ml in case of API and placebo spiked with Tilorone.

Peroxide degradation:

API, placebo and placebo spiked with Tilorone were subjected to sonication with 15ml of hydrogen peroxide at 0.3% v/v for 48 hours at 80°C. Samples subjected to stress were cooled and diluted with a final concentration of 0.195mg/ml in a case of API and a placebo spiked with Tilorone.

Thermal degradation:

API, placebo and placebo spiked with Tilorone were weighed separately in standard volumetric flasks, capped and stored in a hot air oven at 80°C for 18 hours. The stressed sample was cooled and dissolved with diluent for a final concentration of 0.95mg/ml in case of API and placebo spiked with Tilorone.

Method Validation:

Accuracy:

The accuracy solutions were prepared in the triplet of LOQ at 150% by adding Tilorone and impurities (TLHC01 and TLHC02). Accuracy is calculated using aggregate impurity with respect to recovered impurities. The recovery of Tilorone, impurities (TLHC01 and TLHC02) was determined.

Precision:

The accuracy of the method was evaluated as repeatability by injecting six individual Tilorone preparations at the target concentration (0.5µg/mL). The percentage of relative standard deviation (% RSD) of six peak area values obtained was calculated.

Detection limit and limit of quantification:

The limit of detection (LOD) and the limit of quantification (LOQ) for Tilorone, TLHC01 and TLHC02 were estimated at signal/noise ratios of 3:1 and 10:1, respectively, injecting a series of solutions diluted with known concentrations.

Linearity:

The linearity test solutions for the method were prepared from a stock solution at six concentrations of the assay analyte (0.157, 0.295, 0.590, 0.983, 1.967, 3.934µg/ mL). Each solution was injected in triplicate and the average peak area with respect to the concentration data was analyzed with linear least squares regression equation. The linearity test solutions of Tilorone, TLHC01 and TLHC02 were prepared by diluting the stock solutions of impurities at the required concentrations. The standard solutions of TLHC01 (0.153,0.229, 0.535, 1.070, 1.834, 3.820µg/mL) and TLHC02 (0.166, 0.248, 0.580, 1.159, 1.987, 4.140µg/ mL) at six concentration levels of the tilorone target concentration. The slope and Y-intercept of the calibration curve were calculated.

RESULTS AND DISCUSSION:

Method development and optimization:

The main objective of the chromatographic method was to appropriately make the retention time of Tilorone and to separate impurities, TLHC01 and TLHC02 from the analyte peak. Several stationary phases and mobile phases have been optimized with buffers, such as phosphate and acetate with different pH values (2-6) and organic modifiers, in the mobile phase, including acetonitrile and methanol.

For the initial test, an ammonium acetate buffer with a pH value of 6.0 and methanol (50:50, v / v) was chosen at a flow rate of 1.0 mL/min with an ID column of 250 mm X 4.6 mm and a particle size of 5mm with stationary phase C₁₈. Taking into account the high concentration of the acetates and their damage to the column, the volatile mobile phases were studied and satisfactory results were obtained with the mobile phase consisting of the mobile phase A: 20mM potassium dihydro phosphate + 2ml of triethylamine, pH 2.30 and mobile phase B: acetonitrile, methanol and water 60: 20: 20% v / v. A gradient program was followed [Table 1] for 55 minutes.

Table 1: Optimized Gradient Programme

Time (Min)	% Mobile phase - A 20mM Potassium di hydrogen phosphate + 2mL Triethylamine, pH 2.30	% Mobile phase - B Acetonitrile, Methanol and Water 60:20:20%v/v
0	78	22
5	78	22
20	62	38
30	45	55
35	45	55
40	15	85
41	15	85
46	78	22
52	78	22

Chromatographic separation was performed on a Zorbax SB-phenyl [150mm X 4.6mm, 3μ]. The flow rate was 1 ml/minute and the sample injection volume was 10μl. The column temperature was maintained at 40°C. The detection wavelength was set at 269nm. A mixture of buffer and methanol in the proportion of 70: 30% v / v is used as a diluent in the preparation of analysis solutions.

Method validation:

The method has been validated to demonstrate compliance with regulatory requirements. The guidelines of the International Conference on Harmonization for the Validation of Analytical Procedures have been followed: text and methodology: Q2 [R1].¹³

System suitability:

The system suitability test was carried out to verify that the analytical system worked as desired and that it could provide precise and accurate results. The diluted standard and resolution solution were injected five times into the HPLC system. The results are shown in Table 2. All values were found within acceptable limits. Figure 2a-f represents the chromatograms of blank, placebo, tilorone and their official impurities (TLHC01 and TLHC02) respectively.

Figure 2: (a) Blank Chromatogram; (b) Placebo chromatogram; (c) Chromatogram of Tilorone; (d) Chromatogram of impurity TLHC01; (e) Chromatogram of impurity TLHC02; (f) A typical Chromatogram spiked with Tilorone, impurity TLHC01 and TLHC01

Table 2: System suitability data

S. No	Parameter*	Tilorone	TLHC01	TLHC02
1.	Rt (min)	12.635	33.704	20.484
2.	RRT	--	1.80	2.65
3.	%RSD of Peak Area	1.45	1.81	1.02
4.	Tailing factor	1.12	0.95	1.16
* Average of 6 replicates

Figure 3: Degradation Chromatogram of (a) Acid (b) Base (c) Peroxide (d) Heat

Table 3: Forced Degradation and mapping of official impurities in Tilorone

Stress condition	% Assay	% Degradation of Telorene	% Impurities	Mass balance	Peak purity index	Peak purity result
Control	98.65	--	3.50	100.01	0.9999	Pass
Acid	84.21	15.79	21.54	99.56	1.0001	Pass
Base	79.85	20.15	15.32	99.24	0.9999	Pass
Peroxide	89.24	10.76	18.23	101.57	0.9999	Pass
Heat	71.48	28.52	25.14	104.26	0.9997	Pass

Table 4: Accuracy of Tilorone in Related Substances Method

Conc. Level	TLHC01			TLHC02
Conc. Level	Amount added	Amount found	% Recovery	Amount added	Amount found	% Recovery
LOQ*	0.075	0.083	110.7	0.082	0.090	109.8
100%*	0.754	0.683	90.6	0.824	0.754	91.7
150%*	1.131	1.256	111.1	1.233	1.361	110.4

*Average of three replicates (n=3)

Table 6: Results of Linearity

S. No	Tilorone		TLHC01		TLHC02
S. No	Concentration (µg/ml)	Area	Concentration (µg/ml)	Area	Concentration (µg/ml)	Area
1	0.157	12194	0.153	28934	0.166	26364
2	0.295	25929	0.229	43465	0.248	39724
3	0.590	48231	0.535	101284	0.580	92389
4	0.983	80725	1.070	203663	1.159	184896
5	1.967	153951	1.834	350119	1.987	316520
6	3.934	325460	3.820	730771	4.140	659803
Y – intercept		-809.23	-755.59		40.39
Correlation Coefficient (r)		0.999	0.999		0.999
Bias (%)		0.96	-1.00		0.43

Accuracy:

Accuracy solution was prepared in triplicate from LOQ to 150% by spiking Tilorone with Impurities (TLHC01 and TLHC02) of its impurity limits concentration (i.e., Tilorone 0.59µg/ml, TLHC01 0.53µg/ml and TLHC02 0.58µg/ml). Accuracy is calculated by impurity added verses impurities to recover. Recovery for Tilorone and Impurities (TLHC01 and TLHC02) is found within 80% to 120%; hence, the method is accurate. The accuracy data of the method is presented in Table 4.

Precision:

It was performed by carrying out the analysis of the six homogenous solutions of the same test sample and the content of impurities was calculated. The determinations were carried out one after the other under conditions as similar as possible. The relative standard deviation was calculated from the results of the obtained observations. The %RSD of the content of Impurities and total impurities was found to be less than 10% for all the six homogenous test solutions, hence the method is precise. Results of Precision were given in table 5.

Detection limit [LOD] and limit of quantification [LOQ]:

LOQ and LOD were established by determining the signal-to-noise ratio. The experiment was conducted from separate chromatography (diluent and placebo) and placebo spiked with TLHC01 and TLHC02. The detection limits for tilorone and impurity TLHC01 and TLHC02 were 0.023µg/ml, 0.057µg/ml and 0.019µg/ml respectively and the quantification limits were 7.422µg/ ml, 5.857 and 6.396µg/ml respectively.

Table 5: Precision data of the method

S. No	% Related compound
S. No	TLHC01	TLHC02
1	0.182	0.201
2	0.180	0.199
3	0.182	0.200
4	0.175	0.195
5	0.179	0.205
6	0.185	0.21
Average	0.181	0.202
STDEV	0.003	0.005
%RSD	1.879	2.580
Limit	Not more than 10%

Linearity:

Linearity was determined by linear regression analysis. Linearity was obtained for the LOQ tilorone at a sample concentration of 150% w/w, ie 0.157 - 3.934μg/ml and for the LOQ impurities TLHC01 and TLHC02 at 150% w/p specification level, ie., for TLHC01 it is between 0.153 and 3.820μg/ml and for TLHC02 it is between 0.166 and 4.140μg/ml. In all cases, it was found that the regression coefficient was not less than 0.99. The details of the calibration table are shown in Table 4.

CONCLUSION:

In this work, sensitive, specific and reproducible stability-indicating HPLC method for the quantification of the degradants and impurities related to the process with Tilorone tablets was established. The need to develop an analytical method was identified due to the inadequate capacity of the HPLC methods reported to resolve between known impurities and placebo peaks. The developed method shows a good separation and resolution between known impurities, degradation impurities and impurities related to the process with Tilorone tablets. The method was validated according to the ICH guidelines for specificity, linearity, accuracy and precision, the limit of quantification and limit of detection. The results show that the method is suitable for evaluating the stability of Tilorone Tablet.

ACKNOWLEDGMENT:

The authors would like to thank the management of Santhiram College of Pharmacy, Nandyal, Kurnool Dist. A. P. for providing the infrastructure to do this work. All authors discussed the results and contributed to the final manuscript.

CONFLICTS OF INTEREST:

The authors declare that they have no conflicts of interest.

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Received on 08.11.2019 Modified on 06.12.2020

Research J. Pharm. and Tech. 2021; 14(6):3319-3324.

DOI: 10.52711/0974-360X.2021.00577