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.

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

  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.

Table 2: Method optimization.

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

  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.

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).

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

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

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

Table 7: Robustness study.

  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.

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

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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.