Stability Indicating HPTLC Determination of Terazosin in Tablet Formulation

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2. Material and Method

Terazosin was provided by Noche Labs Private Limited, Hyderabad and all the solvents purchased from Merke Limited, Mumbai & Thomas Baker, Mumbai and used such. The samples were spotted in the form of bands of width 6mm with a Camag 100microlitre sample (Hamilton, Bonaduz, Switzerland) syringe on silica gel precoated aluminum plate 60F-254 plates, [20cm × 10cm with 250μm thickness; E. Merck, Darmstadt, Germany)] using a Camag Linomat V (Switzerland) sample applicator. Both test and standard samples (5μL each) were applied onto the HPTLC plate as 6mm wide bands and 12mm apart from middle of bands by spray-on technique along with nitrogen gas supply for simultaneous drying of bands, by means of a Camag (Switzerland) Linomat V sample applicator fitted with a 100μL syringe (Hamilton, Bonaduz, Switzerland). Wavelength selected was 254nm.

Figure 2. Calibration curve of terazosin

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2.1. Preparation of Standard Stock Solution

Reference standard of Terazosin (100mg) was transferred to 10ml volumetric flask and dissolved in methanol:water (1:1). The flask was shaken for 10min and the volume was made up to the mark with the same solvent to obtain standard stock solution of Terazosin (10000μg/ml; stock solution). The working standard solution of Terazosin was prepared from suitable aliquots of stock solution were pipette out and volumes were made up to the mark with mobile phase.

2.2. Preparation of Calibration Curve

From standard stock solution, aliquots are made with diluents to obtain concentration of 100-2500µg/ml of Terazosin. The solution of (20µl) was spotted into plate with the help of syringe. All measurements were repeated three times for each concentration. The calibration curves of the area under curve Vs concentration were recorded for terazosin.

2.3. Validation
2.3.1. Specificity

The acid decomposition study was performed by refluxing the solution of drug in 0.1 N HCl solutions at 40°C for 2 days. For alkaline hydrolysis the solution of drug was refluxed in 0.1 N NaOH solution at 40°C for 10hours. For oxidative decomposition study was performed by exposing the solution of drug in 30% H₂O₂ (v/v) solution at 40°C up to 5 days. The photolytic degradation was performed by exposing the solution of drug to UV light for upto 5 days. Lastly, thermal degradation was performed by standing the pure drug in solid form in stability chamber at 40°C and 75% RH for upto 5 days. All of the solutions were filtered through whatman filter paper and properly diluted to prepare 500µg/ml concentration solution. Samples were then runned under optimized chromatographic condition.

2.3.2. Linearity

This parameter was assessed by correlation coefficient, by visualizing calibration curve and plot of residuals.

2.3.3. Precision

Repeatability was assessed by running six times 500µg/ml solution at a time. For interday and intraday precision was assessed by using 400, 500 and 600µg/ml dilutions

2.3.4. Accuracy

Powdered Terapress (Intas Pharmaceuticals) equivalent to 10mg terazosin was transferred to 10ml volumetric flask and ultrasonication was done for 15minutes with approximately 5ml methanol:water (1:1). Solution was then diluted up to the mark with same solvents and filtered through whatman filter paper. 2ml of this solution was spiked in three different 10ml volumetric flask with 2, 3, and 4ml previously analyzed standard stock solution. Finally volume was made up to the mark with methanol:water (1:1) and estimation of drug content was done by proposed method.

Recovery was investigated for three QC levels (400, 500, and 600) for terazosin.

2.3.5. Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ were calculated through standard deviation of intercept of the calibration curve (28). Limit of detection and quantitation were calculated by using following equation.

2.3.6. Robustness

The robustness of the method was evaluated by the changing the experimental condition such as mobile phase composition, amount of mobile phase and time from spotting to chromatography etc.

Following parameter were considered during the course of robustness study:

A: Mobile phase composition change

B: Amount of mobile phase change

C: Time from spotting to chromatography change.

3. Results and Discussion

3.1. Optimization of Mobile Phase

Mobile phase was optimized by varying proportions of methanol, toluene and chloroform. It was found that mobile phase composition Chloroform:Toluene:MeOH (9:1:6) gives sharp peak at Rf ≈ 8. Chromatogram of optimized chromatographic condition is presented under Figure 3.

Figure 3. Chromatogram of optimized chromatographic condition

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3.2. Validation
3.2.1. Specificity

Specificity of the stability indicating method was established by separation of the principle peak with degradants during forced degradation. The stress conditions utilized were acid hydrolysis, alkaline hydrolysis, oxidation by peroxide, thermal degradation and photo degradation by UV light. No degradation was observed by thermal degradation. Degradants were found to be properly separated by parent peak of terazosin in each case. Product obtained by acidic and alkaline degradation was 2-piperazinyl-6,7-dimethoxy-4-aminoquinazoline confirmed by LC-MS studies and a mass of 290.4 was obtained in positive ESI mode (Bakshi et al.). Terazosin forms tetrahydrofuroic acid, which decomposes further to tetrahydrofuran and carbon dioxide (26). Chromatograms related to stress studies are presented under Figure 4, Figure 5, Figure 6.

Figure 4. Chromatogram of acidic hydrolysis

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Figure 5. Chromatogram of alkaline hydrolysis

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Figure 6. Chromatogram of degradation by UV light

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This is clear in the study that all of the potential interferents are well separated from the parent peak proves method sufficient specific for the determination of Terazosin in pharmaceutical formulations.

Figure 7. Possible degradation pathway of terazosin (i) 2-piperazinyl-6,7-dimethoxy-4-aminoquinazoline (ii) tetrahydrofuroic acid (iii) tetrahydrofuran

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3.2.2. Linearity

Correlation coefficient of 0.999 shows good linearity. By visualizing calibration curve (Figure 2) this is clear that all of the points are closed to regression line also proves linearity of the method. The residuals are distributed both sides equally confirming the linearity of the proposed method.

Figure 8. Plot of residuals

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3.2.3. Precision

Repeatability RSD was found to be 0.331. Intraday and interday precision RSD was found to be between 0.337-0.486 and 0.378-0.885 respectively.

Values (RSD) in each case found was less than 1, shows method is precise in nature.

3.2.4. Accuracy

Accuracy studies were performed on terapress tablets. Results are presented under Table 1.

Table 1. Recovery study of Terazosin

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Percentage recovery found was between 99.15-100.64% proves method to be accurate.

3.2.5. Limit of Detection (LOD) and Limit of Quantification (LOQ)

LOD and LOQ of the method were found to be 18.06µg/ml and 54.72µg/ml.

Robustness

In each case there is no significant change is found (as RSD values are lower than 2) shows method sufficient robust for the analysis of terazosin.

Table 2. Data for robustness

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3.3. Analysis in Formulations

The tablet powder equivalent to 2mg terazosin was transferred to a 10 ml volumetric flask and diluted with methanol:water (1:1) to about 50ml. The solution was ultrasonicated for 15 minute and diluted up to the mark with methanol:water (1:1). The solution was then carefully filtered with filter paper before spotting on TLC plate in HPTLC system.

Table 3. Results of analysis of formulation by proposed method

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