Available online on 15.06.2024 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2024 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                            Research Article

Method development and validation of a new stability indicating HPLC and LC-ESI-MS/MS methods for the determination of Tavaborole

¹ Dipak Chandrakant Kulkarni, ¹ Anima Sunil Dadhich, ²* Mukthinuthalapati Mathrusri Annapurna

¹ Department of Chemistry, GITAM School of Science, Visakhapatnam, India

² GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India

 

 

INTRODUCTION

Tavaborole (Figure 1) is an antifungal agent used for the fungal infection of nail and nail bed. FDA has given approval for the treatment of onychomycosis^1-3 in July 2014. Tavaborole (C₇H₆BFO₂; Mo. Wt. 151.93 g/mol) is chemically 5 - Fluoro -1,3 - dihydro -2,1-benzoxaborol -1-ol. It acts by inhibiting an essential fungal enzyme required for protein synthesis. This inhibition of protein enzyme (aminoacyl transfer ribonucleic acid) synthesis leads to cell growth termination and causes death of the cell which finally eliminates the fungal infection. 

Tavaborole was earlier studied by spectrophotometry^4-5 and HPLC^6-7 and in the present study a new stability indicating LC-APCI-MS/MS method has been proposed for the estimation of Tavaborole and the method was validated as per ICH guidelines⁸.

 
 Figure 1: Chemical structure of Tavaborole 

MATERIALS AND METHODS

Tavaborole API was obtained as gift sample from Zydus Lifesciences Ltd (India) and it is available as 5% topical solution from Alembic Pharma, Viona Pharmaceuticals etc. 

Instrumentation

AB SCIEX Instruments Linear Ion Trap Quadrupole LC-MS/MS Mass Spectrometer (Model no. 5068379-Y) QTRAP Enabled Triple Quad 5500+ with ExionLC Binary Gradient AD Pump, Autosampler AD Autosampler, Column Oven AC Column Oven and Agilent column PDA detector was employed for the present study. Agilent ZORBOX C18 (150 mm x 4.6 mm x 3 µm) column was employed and the injection volume was 20 µL and the total run time was 10 mins (Detection wavelength 254 nm). A mixture of 5 mM Ammonium formate: Methanol (30: 70) was used as mobile phase on isocratic mode with 1 ml/min as flow rate. 0.1% Formic acid: Acetonitrile was used as mobile phase on gradient mode with flow rate was 1 ml/min.

MS Conditions

AB SCIEX Instruments 

Triple Quad 5500+ QTRAP

Model                                                                   :5068379-Y

Ionization mode                                          :ESI positive 

Source temp.                                                 :650ºC

Source type                                                       :Turbo spray

Heater gas (GS1)                                            : 60 ºC)                        

Nebulizer gas (GS2)                                      :65 ºC)

Collision energy                                          :35 V 

Preparation of stock solution

25 mg of Tavaborole API was accurately weighed and carefully transferred into a 25 ml volumetric flask and was dissolved in HPLC grade Methanol (1000 µg/ml) and this stock solution was sonicated for 30 mins and then diluted with the diluent as per the requirement.

Method validation⁸

0.5-100 µg/ml Tavaborole solutions were prepared from the stock solution (1000 µg/ml) on dilution with the mobile phase consisting of a mixture of 5 mM Ammonium formate: Methanol (30: 70) and each solution was injected (n=3) into the system and the average peak area from the respective chromatograms was calculated. A calibration graph was drawn by plotting the concentration of the drug solutions on the x-axis and the corresponding peak area of the chromatograms on the y-axis. The intraday precision studies were conducted on the same day at different equal time intervals and the inter-day precision studies were conducted on three successive days (Day 1, Day 2 and Day 3) and the % RSD was calculated. Accuracy studies were performed by spiking the formulation solution with 50%, 100% and 150% API solution and thereby the percentage recovery was calculated with the help of regression equation. The percentage relative standard deviation was calculated in all the validation parameters.

Assay of Tavaborole

Tavaborole is available as topical solution (Label claim: 5%) Alembic Pharmaceuticals Ltd, Viona Pharmaceuticals, Zydus Lifesciences Ltd, Encube Ethicals Pvt. Ltd. in India. Two different brands of Tavaborole were collected and extracted with methanol and after sonication diluted with the mobile phase as per the requirement. The resulting solution was filtered through 0.24 μm membrane filter and 10 μL of these formulation solutions were injected in to the HPLC system. The peak area of the chromatogram (n =3) was noted and the percentage purity was determined.

Stress degradation studies⁹

During the acidic degradation study Tavaborole solution was heated with 0.1N HCl at 70ºC for about 30 mins and neutralized with 0.1N NaOH solution. The contents were diluted with mobile phase and the resultant solution was injected into LC-MS/MS system and the peak area of Tavaborole was noted from the respective chromatogram and the mass spectrum was recorded.

During the acidic degradation study Tavaborole solution was heated with 0.1N NaOH at 70ºC for about 30 mins and neutralized with 0.1N HCl solution. The contents were diluted with mobile phase and the resultant solution was injected into LC-MS/MS system and the peak area of Tavaborole was noted from the respective chromatogram and the mass spectrum was recorded.

During the thermal degradation study Tavaborole solution was heated at 70ºC for about 30 mins and the contents were diluted with mobile phase and the resultant solution was injected into LC-MS/MS system and the peak area of Tavaborole was noted from the respective chromatogram and the mass spectrum was recorded.

During the oxidative degradation study Tavaborole solution was heated with hydrogen peroxide at 70ºC for about 30 mins and then diluted with mobile phase and the resultant solution was injected into LC-MS/MS system and the peak area of Tavaborole was noted from the respective chromatogram and the mass spectrum was recorded.

RESULTS AND DISCUSSION

A new stability-indicating LC-ESI-MS/MS method has been proposed for the estimation of Tavaborole in pharmaceutical formulations using AB SCIEX Instruments LC-ESI-MS/MS (Model no. 5068379-Y) QTRAP Enabled Triple Quad 5500+ with Agilent Zorbox C18 (150 mm x 4.6 mm x 3 µm) column and PDA detector. The total run time was 10 mins and the detection wavelength was 254 nm. A mixture of 5 mM Ammonium formate: Methanol (30: 70) was used as mobile phase on isocratic mode with 1 ml/min as flow rate. A brief review of the reported methods was summarized and some of the parameters were compared with the present proposed method and the details were given in Table 1.

 

 

Table 1: Literature survey

Method	Mobile phase (v/v) / Reagent	λmax (nm)	Linearity (µg/mL)	Ref
Spectrophotometry	Distilled water	272 265	20-100	4
Spectrophotometry	Methanol, Water, HCl, NaOH, Phosphate buffers  (pH 2.0, 4.0 & 7.0)  Borate buffer (pH 9.0)	271	1-100 1-80	5
RP-HPLC	10 mM Phosphoric acid  (pH 2.0): Acetonitrile (70:30)	220	-	6
RP-HPLC (Gradient mode)	Methanol: Acetonitrile (50:50) 0.05% of aq. Perchloric acid: 0.05% Perchloric acid in Acetonitrile & Methanol (50:50)	-	0.05- 4	7
LC-ESI-MS/MS	10 mM Ammonium formate: Methanol (30: 70) (Isocratic mode)	254	0.5-100	Present method

 

 

Tavaborole was eluted at Rt 2.45 min with theoretical plates more than 2000 and tailing factor less than 1.5 and the corresponding chromatograms of blank, Tavaborole  API and the mass spectrum of Tavaborole with the optimized chromatographic conditions were shown in Figure 2.

 

 

 

 

 

Linearity, Precision, Accuracy and Robustness

Tavaborole  obeys Beer-Lambert’s law over the concentration range 0.5-100 µg/ml (Table 2) and the linear regression equation was found to be y = = 3316.9x + 79.061 (R² = 0.9999) Figure 3). The LOD and LOQ values were found to be 0.4129 µg/ml and 1.3721 µg/ml respectively.

The % RSD in intraday precision (0.52), interday precision (0.62-0.82) (Table 3) was found to be less than 2.0% stating that the method is precise. In the accuracy study the % RSD was found to be 0.27-0.72 (<2) (Table 4) with a recovery of 99.58.99-99.92 indicating that the method is accurate.

 

 

 

Table 2: Linearity

*Mean of three replicates

Figure 3: Calibration curve

 

 

 

Table 3: Precision study 

*Mean of three replicates

Table 4: Accuracy study 

*Mean of three replicates

Assay of Tavaborole  

The assay of Tavaborole  topical solution was performed using the proposed method with the optimized conditions and the percentage of purity of Tavaborole  was found to be 98.20-99.20 (Table 5).

Table 5:  Assay of Tavaborole 

*Mean of three replicates

Stress degradation studies

Tavaborole (20 µg/ml) was exposed to different stress conditions under the optimized chromatographic conditions and then injected in to the system.  

During the acidic degradation, Tavaborole  was eluted at Rt 2.45 min and a degradant was eluted at 5.60 mins and about 31.78 % has undergone decomposition. The mass spectra of Tavaborole  and its degradant were shown in Figure 4.

Table 6: Stress degradation studies 

*Mean of three replicates

 

 

During the thermal degradation, Tavaborole was eluted at Rt 2.45 min and no degradants were observed. about 4.53 % has undergone decomposition. The mass spectra of Tavaborole  was shown in Figure 5.

 

During the basic degradation, Tavaborole was eluted at Rt 2.45 min and two degradants were eluted at 2.00 mins and 3.14 mins. about 39.55 % has undergone decomposition. The mass spectra of Tavaborole  and its degradants were shown in Figure 6.

 

During the oxidative degradation, Tavaborole  was eluted at Rt 2.45 min and a degradant was eluted at 1.43 mins. about 39.55 % has undergone decomposition. The mass spectra of Tavaborole  and its degradant were shown in Figure 7.

 

 

The details of the stress degradation studies of Tavaborole  were shown in Table 6. It is observed that Tavaborole  is highly sensitive towards the proposed acidic (31.78 %), alkaline (25.75 %) and oxidative conditions (39.55 %) and less than 5% degradation was observed during thermal degradation (4.53%).

CONCLUSION

The authors have developed a new stability indicating LC-ESI-MS/MS method for the estimation of Tavaborole  and the method is simple, precise and accurate and used for the routine analysis of Tavaborole  in pharmaceutical formulations and no interference of excipients was observed during the assay.

Conflicts of Interest: The authors declare no conflict of interest.

Acknowledgement: The authors are grateful to Zydus Lifesciences Ltd (India) for providing the gift samples of Tavaborole and the authors declare no conflict of interest.

REFERENCES

1. Markham A, “Tavaborole: First global approval” Drugs, 2014; 74(13):1555-1558. https://doi.org/10.1007/s40265-014-0276-7
    PMid:25118637
2. Boni EE, Raza Aly, Sheryl LB et al., “Efficacy and safety of Tavaborole topical solution, 5%, a novel boron-based antifungal agent, for the treatment of toenail onychomycosis: Results from 2 randomized phase-III studies” Journal of the American Academy of Dermatology, 2015; 73 (1):62-69. https://doi.org/10.1016/j.jaad.2015.04.010 PMid:25956661
3. Toledo-Bahena ME, Bucko A, Ocampo-Candiani J, Herz-Ruelas ME, Jones TM, Jarratt MT, Pollak RA, Zane LT, “The efficacy and safety of Tavaborole, a novel, boron-based pharmaceutical agent: Phase 2 studies conducted for the topical treatment of toenail onychomycosis” Journal of Drugs Dermatology, 2014; 13(9):1124-1132.
4. Anas HI, Sagir UA, Salman MY, Amar GZ and Vishal SG, “UV-spectrometric method development and validation of Tavaborole” Journal of Chemical and Pharmaceutical Research, 2021; 13(5):1-9.
5. Sai Sheela A, Mathrusri Annapurna M and Spandana Yasaswini R, “New validated analytical methods for the determination of Tavaborole (An anti-fungal agent)” Research Journal of Pharmacy and Technology, 2020; 13(4):1895-1900. https://doi.org/10.5958/0974-360X.2020.00341.8
6. Tampucci Silvia, Terreni Eleonora, Burgalassi Susi, Chetoni Patrizia, Monti Daniela, “Development and validation of an HPLC-UV Method to quantify Tavaborole during in vitro transungual permeation studies” Journal of AOAC International, 2018; 101(2):437-443. https://doi.org/10.5740/jaoacint.17-0039 PMid:28766480
7. Siva Rao T, Venu Balireddi and Krishna Murthy T, “Analytical method development and validation of stability indicating method of RP-HPLC for quantification of Tavaborole related substances: Application to 5% topical solution” European Journal of Biomedical and Pharmaceutical Sciences, 2018; 5(9):545-550.
8. ICH Validation of analytical procedures: Text and methodology Q2 (R1), International Conference on Harmonization, 2005.
9. ICH Stability testing of new drug substances and products Q1A (R2), International Conference on Harmonization, 2003.