Acta Scientific Pharmaceutical Sciences (ASPS)(ISSN: 2581-5423)

Research Article Volume 5 Issue 8

Development and Validation of a New Reverse Phase Liquid Chromatographic Method for the Assay of Tilorone

Yenda Manishankar* and Mukthinuthalapati Mathrusri Annapurna

Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India

*Corresponding Author: Yenda Manishankar, Department of Pharmaceutical Analysis and Quality Assurance, GITAM Institute of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.

Received: May 22, 2021; Published: July 21, 2021

Abstract

  A new RP-HPLC method has been proposed for the assay of Tilorone in API and its tablet dosage forms. Tilorone is an anti-viral immune stimulating medication. It can be used for the treatment of viral hepatitis A, B, C, urogenital tract and respiratory infections. It stimulates the formation of α-, β-, γ-interferons and produces interferon intestinal epithelial cells, neutrophils, T-lymphocytes and hepatocytes in the body. A mobile phase composition consisting of tetra butyl ammonium hydrogen sulphate and acetonitrile was chosen for the chromatographic study on isocratic mode. C8 Agilent column has been used for the chromatographic elution of Tilorone and the HPLC system was monitored at 264 nm with flow rate 0.5 mL/min. Tilorone has shown linearity over the concentration range 0.05-40 µg/mL and the regression equation was y = 428331x + 24602 with correlation coefficient 0.9999. The LOD and LOQ are found to be 0.0138 and 0.0429 µg/mL respectively. The method was validated as per ICH guidelines. The proposed RP-HPLC method was found to be precise, accurate, and robust for the quantification of Tilorone in pharmaceutical dosage forms.

Keywords: RP-HPLC; Tilorone; Acetonitrile; ICH Guidelines

Introduction

  Tilorone (CAS 0027591-97-is an effective anti-viral agent against influenza viruses, herpesviruses, hepato viruses etc. Tilorone hydrochloride (Figure 1) is a synthetic orally active interferon inducer which has anti-cancer as well as anti-inflammatory activities. The antiviral action of Tilorone is associated with the inhibition of translation of virus-specific proteins followed by the suppression of virus replication. Tilorone is a fluoren-9-ones derivative and it is an alpha 7-nicotinic aceetyl choline receptor agonist. It has a molecular formula C24H34Cl2N2O3. 2HCl and molecular weight 483.47. Very few analytical methods were so far developed in the literature HPLC/MS/MS methods were developed by Zhang., et al. [2] and Xianhua., et al. [3] for the simultaneous quantification of Tiloronoxim and Tilorone in human urine in presence of an internal standard, Metoprolol and also in human blood respectively. Tilorone is an active major metabolite of Tiloronoxim. One spectrophotometric [4] method and one RP-UFLC [5] method were reported in the literature for the assay of Tilorone in pharmaceutical dosage forms. At present the authors proposed a new reverse phase liquid chromatographic method (RP-HPLC) for the quantification of Tilorone in tablets and the method were validated as per ICH guidelines [6].

Figure 1: Clinical photography of nodular bony graft after FDP removal and extraction of both of lower left second premolar and second molar.

Materials and Methods

Instrumentation and Chemicals

  Acetonitrile (HPLC grade) and tetra butyl ammonium hydrogen sulphate (TBHS) were procured from Merck (India). Shimadzu Model HPLC system with C8 Agilent column and photodiode array detector were used for the chromatographic analysis. Tilorone was supplied by HONOUR Labs Limited (Hyderabad, India) as gift sample. Tilorone is available with brand names AMIXIN and LAVOMAX (Label claim: 60 mg) as film coated tablets.

Preparation of tetra butyl ammonium hydrogen sulphate solution (pH 3.5)

  The molecular weight of tetra butyl ammonium hydrogen sulphateis 339.5 grams/mole and it is an ion pairing reagent. 10 mM solution of tetra butyl ammonium hydrogen sulphate was prepared by dissolving accurately 3.395 grams in HPLC grade water in a 1000 ml volumetric flask and the solution was sonicated, filtered through a membrane filter and then used as mobile phase.

Preparation of Tilorone stock (1000 μg/mL) solution

  25 mg of Tilorone was accurately weighed and dissolved in a 25 mL volumetric flask and made up to the volume with acetonitrile (HPLC grade) to obtain 1000 μg/mL. This stock solution was diluted as per the requirement with the diluent and filtered through membrane filter before use. The diluent used was TBHS: Acetonitrile (50:50, v/v).

Method validation [6] Linearity study

  A series of solutions (0.05- 40 µg/mL) were prepared from the stock solution and diluted with mobile phase.20 µL of each of these solutions were injected in to the HPLC system and the peak area of each chromatogram was noted. A calibration curve was drawn by plotting the concentration on the x-axis and the corresponding mean peak area values on the y-axis.

Precision study

  Precision study was performed on the same day with three different concentrations and on different days using three different concentrations which are called as Intraday and Inter-day precision studies respectively. Three different concentrations 5, 10 and 20 µg/mL of Tilorone solutions were prepared for intraday and interday precision studies and each solution was injected three times (n=3) on the same day and on three consecutive days and the average peak area was calculated from the individual chromatograms obtained.

Accuracy study

  The accuracy study was performed using the standard addition method. In this study the recovery values and the % RSD values were calculated. In this method 50%, 100% and 150% of the pure drug (API) solutions were added to a fixed concentration of the extracted formulation solution and 20 µL of each of these resultant solutions were injected (n = 3) in to the HPLC system and the peak area of each chromatogram was noted. The mean peak area was calculated and there by the percentage recovery was calculated using the linear regression equation obtained in the linearity study.

Robustness study

  Robustness is one of the important validated parameters and in this study small changes such as flow rate (± 0.1mL/min), mobile phase ratio (± 2%), pH and detection wave length (259 nm and 269 nm) were incorporated in the optimized method and 20 µL of 10 µg/ml of solution was injected (n = 3) in to the HPLC system after attaining the base line with the incorporated changes one by one and the peak area of each chromatogram obtained was noted. The mean peak area of the chromatograms obtained from each incorporated change was calculated and there by the percentage recovery was also calculated using the linear regression equation obtained in the linearity study.

Assay of commercial formulation (Tablets)

  20 tablets of two different brands of Tilorone available in pharmacy store were purchased, weighed powdered. Powder equivalent to 25 mg Tilorone was weighed accurately and extracted with HPLC grade acetonitrile. The extracted solution was further diluted with the mobile phase, sonicated for half an hour and filtered through 0.45 mm membrane filter before injecting in to the HPLC system to prevent the particulate matter if any. 20 µL of these solutions were injected (n = 3) in to the HPLC system and the mean peak area was noted from the respective chromatograms. The percentage of purity was calculated by substituting the mean peak area value in the linear regression equation.

Results and Discussion

  A new reverse phase liquid chromatographic method has been developed for the determination of Tilorone in pharmaceutical dosage forms using Shimadzu Model HPLC system with C8 Agilent column and photo diode array detector. The previously published analytical methods were compared with the present proposed method in table 1. Mobile phase mixture consisting of tetra butyl ammonium hydrogen sulphate and acetonitrile was selected for the chromatographic study. Tetra butyl ammonium hydrogen sulphate is an ion pairing reagent. The run time was 10 minutes and the detection wavelength was at 264 nm. The chromatographic study was performed isocratic mode with a flow rate of 0.5 mL/min.

Method

Mobile phase

(% v/v)

Linearity

(µg/mL)

Comments

Ref

HPLC–MS/MS

Metoprolol

(Internal standard)

Methanol: 15 mMAmmonium bicarbonate (pH 10.5)

0.001-0.1

Human urine

Gradient mode

 

[2]

HPLC–MS/MS

Methanol: 15 mMAmmonium bicarbonate (pH 10.5)

0.001-0.1

Human Blood

Gradient mode

[3]

Spectrophotometry

Sodium acetate buffer (pH 4) Borate buffer (pH 9.0)

Phosphate buffer (pH 2.0) Phosphate buffer (pH 5.0)

0.4-14

Good linearity

[4]

RP-UFLC

0.1% TEA: Acetonitrile

(pH 3.2 adjusted with OPA)/(40:60)

0.1-20

Stability indicating (PDA)

[5]

RP-HPLC

Tetra butyl ammonium hydrogen sulphate: Acetonitrile (61: 39)

0.05-40

Ion pairing agent

Present method

Table 1: Comparison of previously published methods with the present method.

Method development and optimization

  Initially a mobile phase consisting of tetra butyl ammonium hydrogen sulphate and methanol (20: 80) was chosen with flow rate 1.0 ml/min for the chromatographic study and Tilorone was eluted at 1.307 mins. Therefore methanol in the mobile phase was replaced with acetonitrile and the ratio was maintained at 50: 50 with flow rate 0.8 ml/min. Tilorone was eluted at 1.466 min with theoretical plates less than 2000. Later the flow rate was decreased to 0.5 ml/min and the retention time of Tilorone was shifted to 1.917 min with theoretical plates more than 2000. To shift the retention time the organic phase was decreased and finally Tilorone was eluted at 2.338 mins and the system suitability parameters were satisfied. The method was optimised with mobile phase, TBHS: Acetonitrile (61:39, v/v) with flow rate 0.5 ml/min and the detection was carried out at 264 nm. The trials runs obtained during the optimization process were shown in Figure 2 and the summary of details was given in table 2.

Trial

Mobile phase (v/v)

Flow rate

(mL/min)

Rt

(min)

Theoretical plates

Tailing factor

Comment

1

TBHS: Methanol (20:80)

1.0

1.307

1631.166

3.424

Tailing factor >2

Theoretical plates <2000

Rt less than 2 min

2

TBHS: Acetonitrile (50:50)

0.8

1.466

1934.812

1.639

Theoretical plates <2000

Rt less than 2 min

3

TBHS: Acetonitrile (50:50)

0.5

1.917

2277.787

1.591

Rt less than 2 min

4

TBHS: Acetonitrile (61:39)

0.5

2.338

2320.445

1.212

Method optimized

Table 2: Method optimization.

Figure 2: Chromatograms obtained during the method optimization of Tilorone (10 µg/ml).

Method validation

  The proposed method was validated by linearity, precision, accuracy, robustness as per the ICH guidelines for the determination of Tilorone. Tilorone has shown linearity over the concentration range 0.05-40 µg/mL (Table 3) and the regression equation was y = 428331x + 24602with correlation coefficient 0.9999. The LOD and LOQ are found to be 0.0138 and 0.0429 µg/mL respectively. The calibration curve was shown in figure 3. The representative chromatograms obtained for the placebo and that of Tilorone standard (API) were shown in Figure 4. The system suitability parameters are within the acceptable criteria.

Conc. (µg/mL)

*Mean peak area

%RSD

Theoretical plates

Tailing factor

0.05

24337

0.39

2498.226

0.979

0.1

47923

0.23

2564.332

0.867

0.5

232921

0.48

2778.329

1.221

1

454256

0.32

2337.005

1.463

2

873387

0.41

2464.754

1.301

5

2231224

0.29

2332.290

1.495

10

4403233

0.34

2302.556

1.374

20

8455549

0.31

2569.534

1.290

30

12943257

0.49

2645.189

1.392

40

17142681

0.52

2561.267

1.269

Table 3: Linearity study.
*Mean of three replicates.

Figure 3: Calibration curve of Tilorone.

  Intraday and inter-day precision were studied and the % RSD was found to be 0.21-0.33 and 0.38-0.59 respectively (<2.0 %) showing that the method is precise (Table 4 and Table 5). The % RSD in accuracy was found to be 0.73-0.91 (<2.0 %) with a recovery of 99.33-99.47 %showing that the method is accurate (Table 6). In robustness study the % RSD was found to be 0.27-1.23 which is less than 2.0 indicating that the method is robust (Table 7).

Conc.

(µg/mL)

Peak area (AUC)

Statistical Analysis

*Mean peak area ± SD (% RSD)

5

2231321

2231306 ± 7363.31 (0.33)

5

2231296

5

2231301

10

4423215

4424190 ± 9290.80 (0.21)

10

4424136

10

4425219

20

8456024

 

8455876.67 ± 2830.87 (0.27)

20

8455933

20

8455673

Table 4: Intraday precision study.
*Mean of three replicates.

Conc.

(µg/mL)

*Mean peak area

*Mean ± SD(% RSD)

Day 1

Day 2

Day 3

5

2302562

2283012

2292261

2292611.67 ± 9628.97 (0.42)

10

4510022

4511214

4510354

4510530 ± 26612.13 (0.59)

20

8362533

8359847

8358475

8360285 ± 31769.08 (0.38)

Table 5: Interday precision study.
*Mean of three replicates.

Conc (µg/mL)

*Average conc.

(% RSD)

% Recovery

Formulation

Pure drug

Total

6

3

9

8.94 (0.89)

99.33

6

3

9

6

3

9

6

6

12

11.93 (0.73)

99.41

6

6

12

6

6

12

6

9

15

14.92 (0.91)

99.47

6

9

15

6

9

15

Table 6: Accuracy study.
*Mean of three replicates.

Parameter

Condition

*Mean peak area ± SD

(% RSD)

Flow rate (± 0.1 ml/min)

0.4

4415984 ± 44601.44 (1.01)

0.5

0.6

Detection wavelength (± 2 nm)

 

259

4404521 ± 11892.21 (0.27)

264

269

Mobile phase composition

TBHS: Acetonitrile (± 2 %, v/v)

56: 44

4405269 ± 25991.09 (0.59)

61:39

66:34

pH (± 0.1 unit)

3.4

4410256 ± 54246.15 (1.23)

3.5

3.6

Table 7: Robustness study.

Assay of tilorone tablets

  The analytical method i.e. RP-HPLC method developed for the determination of Tilorone was validated and it was applied for the assay of marketed formulations. The percentage of recovery was found to be 99.82- 99.88 (Table 8). The typical chromatogram obtained for the marketed formulations were shown in figure 4.

Formulation

Label claim (mg)

*Amount found (mg)

*Recovery (%)

Brand I

60

59.93

99.88

Brand II

60

59.89

99.82

Table 8: Assay of Tilorone.
*Mean of three replicates.

Figure 4: Representative chromatograms of Tilorone.

Conclusion

  The proposed RP-HPLC method was validated as per ICH guidelines and can be used for the determination of Tilorone in pharmaceutical dosage forms. This method is also useful for performing the pharmacokinetic studies and also for the assay of Tilorone in biological samples.

Acknowledgement

The authors are grateful to HONOUR Labs Limited (Hyderabad, India) for supplying Tilorone as gift sample. There is no conflict of interest.

References

  1. Stringfellow D and Glasgow L. “Tilorone hydrochloride: An oral interferon-inducing agent”. Antimicrobial Agents Chemotherapy2 (1972): 73-78.
  2. Zhang X., et al. “Simultaneous quantification of Tiloronoxim and Tilorone in human urine by liquid chromatography-tandem mass spectrometry”. Journal of Chromatography B 2 (2008): 349-357.
  3. Xianhua Zhanga., et al. “Performance of Tiloronoxim and Tilorone determination in human blood by HPLC-MS/MS: Method validation, uncertainty assessment and its application to a pharmacokinetic study”. Journal of Chromatography B 3-4 (2010): 492-496.
  4. Mathrusri Annapurna M and Sri Valli D. “Derivative spectrophotometric methods for the determination of Tilorone - An Anti-retroviral Drug”. Asian Journal of Pharmaceutics1 (2018): S 288-S 295.
  5. Hima Bindu G., et al. “New stability indicating ultrafast liquid chromatographic method for the determination of Tilorone in tablets”. Research Journal of Pharmacy and Technology9 (2018): 3950-3956.
  6. ICH validation of analytical procedures: text and methodology Q2 (R1), International Conference on Harmonization (2005).

Citation

Citation: Yenda Manishankar and Mukthinuthalapati Mathrusri Annapurna. “Development and Validation of a New Reverse Phase Liquid Chromatographic Method for the Assay of Tilorone". Acta Scientific Pharmaceutical Sciences 5.8 (2021): 59-65.

Copyright

Copyright: © 2021 Yenda Manishankar and Mukthinuthalapati Mathrusri Annapurna. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.




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