INTRODUCTION:

Tolterodine Tartrate is described chemically as 2-(3-(diisopropylamino)-1- phenylpropyl)-4-methylphenol Tartrate. Tolterodine is an antimuscarinic drug which is used to treat urinary incontinence. Urinary incontinence is characterized by uninhibited contractions causing an uncontrollable urge for urine. Tolterodine acts on M1, M2, M3, M4 and M5 subtypes of muscarinic receptors whereas older antimuscarinic agents used for overactive bladder acts only on M3 muscarinic receptor^1-3. Tolterodine Tartrate is used for the treatment of urinary urge incontinence and other symptoms of overactive bladder. Tolterodine Tartrate is a potent muscarinic receptor antagonist that is equipotent to oxybutynin in the bladder, but less potent in salivary glands, with the aim of improving tolerability (less dry mouth) in patients with overactive bladder^4,5. Both urinary bladder contraction and salivation are mediated via cholinergic muscarinic receptors.

After oral administration, tolterodine is metabolized in the liver, resulting in the formation of the 5-hydroxymethyl derivative, a major pharmacologically active metabolite⁶. The molecular formula of Tolterodine is C₂₂H₃₁NO. The molecular mass of Tolterodine is 325.488 g/mol⁸. It is soluble in water, methanol, slightly soluble in ethanol and partially insoluble in toluene.

MATERIALS AND METHODS:

Materials:

Standard gift samples of Tolterodine were provided by Dr Benarji Patrudu, Associate Professor, Gitam University, Hyderabad. All the chemicals and reagents used were of analytical grade.

HPLC Chromatographic Parameters:

Chromatographic separation was performed on The HPLC-PDA system used, consisted shimadzu high performance liquid chromatography with LC- 20AT pump and SPD-20A interfaced with LC solution software, equipped with a reversed phase C18 analytical column of 250 mm x 4.6 mm and particle size 5 µm (X-terra C18) with flow rate of 1.0 mL/min.

An HPLC method was developed for Tolterodine by using photo diode array detector. The Tolterodine and related impurities are separated in Acetonitrile: 0.1% v/v phosphoric acid in Water (35:65, v/v). The absorption maxima for Tolterodine were found at 210 nm. Injection volume was set up to 20µL and column temperature was set to 30˚C. By follow this analytical method conditions, Tolterodine and related impurities were separated. The representative blank and standard chromatograms were showed in Figure.1 and Figure 2.

Figure. 1. Representative chromatogram of diluent

Figure. 2. Representative chromatogram of standard

Method of analysis:

Standard solution preparations:

Weigh and transfer about 100 mg of Tolterodine reference standard into a 100mL volumetric flask dissolve and make up to with diluent. Transfer 5mL of this solution into a 100 mL volumetric flask and make up to the volume with diluent.

Evaluation of system suitability:

Inject blank, followed by standard solution for five times into HPLC and evaluate the chromatogram. The system is suitable for analysis, in standard chromatogram the USP tailing factor determined for Tolterodine peak should not be more than 2.0 and the percentage relative standard deviation of peak area of Tolterodine in the five replicate injections should be not more than 2.0.

Sample solution preparations:

Weigh and transfer about 100 mg of sample into a 100mL volumetric flask dissolve and make up to with diluent. Transfer 5mL of this solution into a 100mL volumetric flask and make up to the volume with diluent.

Procedure:

If the system suitability passes, Inject standard preparation-1 and sample preparations-1 and 2 (Order of injections as specified) and record the chromatograms. Determine the area responses of Tolterodine peak in standard and sample preparations. The expected retention time of Tolterodine peak is about 10.0 minutes.

Degradation Study:

Analyze the impurities and Tolterodine individually as per the method to verify the retention times. In order to assess the stability indicating nature of the HPLC method, Tolterodine samples will be stressed by acid, base, hydrogen peroxide, heat and UV radiation. The degraded samples will be analyzed using a PDA detector for determining the peak purity.

Diluent: Acetonitrile: Water (50:50, v/v). The solution was injected (blank).

Test solution:

Weigh and transfer about 100 mg of sample into a 100mL volumetric flask dissolve and make up to the volume with diluent. Transfer 5mL of solution in to a 100mL volumetric flask dissolve and make up to the volume with diluent.

Acid Hydrolysis:

Exposure conditions	At room temperature	At 60˚C temperature
Acid Hydrolysis	Weigh and transfer about 100 mg of sample into a Quality Control 100mL volumetric flask, add 20 mL of diluent and add 2.0 mL of 1.0 N hydrochloric acid solution. Keep the solution at room temperature for three hours and then neutralize with 2.0 mL of 1.0 N sodium hydroxide solution and dilute to 100mL with the diluent. Further dilute to 5mL of solution into100mL volumetric flask and dilute to the volume with the diluent.	Weigh and transfer about 100mg of sample into a 100mL volumetric flask, add 20 mL of diluent and add 2.0 mL of 1.0 N hydrochloric acid solution. Keep the solution at 60°C for three hours and then neutralize with 2.0 mL of 1.0 N sodium hydroxide solution and dilute to 100mL with the diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent.

Base Hydrolysis:

Exposure conditions	At room temperature	At 60˚C temperature
Base Hydrolysis	Weigh and transfer about 100 mg of sample into a 100mL volumetric flask, add 20 mL of diluent and add 2.0 mL of 1.0 N Sodium hydroxide solution. Keep the solution at room temperature for three hours and then neutralize with 2.0 mL of 1.0 N hydrochloric acid solution and dilute to 100mL with the diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent	Weigh and transfer about 100mg of sample into a 100mL volumetric flask, add 20 mL of diluent and add 2.0 mL of 1.0 N Sodium hydroxide solution. Keep the solution at 60°C for three hours and then neutralize with 2.0 mL of 1.0 N hydrochloric acid solution and dilute to 100mL with the diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent.

Oxidation:

Exposure conditions	At room temperature	At 60˚C temperature
Oxidation	Weigh and transfer about 100 mg of sample into a 100mL volumetric flask, add 20 mL of diluent and add 2.0 mL of 5% hydrogen peroxide solution. Keep the solution at room temperature for three hours and dilute to 100mL with the diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent.	Weigh and transfer about 100mg of sample into a 100mL volumetric flask, add 20 mL of diluent and add 2.0 mL of 5% hydrogen peroxide solution. Keep the solution at 60°C for three hours and dilute to 100mL with the diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent.

UV light degradation:

Weigh and transfer about 100mg of substance into a 100mL volumetric flask and dissolve with 20 mL of diluent and expose the solution to UV light at 365 nm for three hours and dilute to 100mL with diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent.

Heat degradation:

Weigh and transfer about 100mg of sample into a 100mL volumetric flask and dissolve with 20 mL of diluent and keep the solution at 60°C temperatures for three hours and dilute to 100mL with the diluent. Further dilute to 5mL of solution into 100mL volumetric flask and dilute to the volume with the diluent.

Solid state stability:

Perform the solid-state stability study by exposing the Tolterodine to heat, white fluorescent light and UV light. Analyze the heat, white fluorescent light and UV light exposed samples along with control sample using a photodiode-array detector for determining the peak purity.

Exposure and sample preparations:

Preparation of Control sample:

Weigh and transfer about 100mg of sample into a 100mL of volumetric flask dissolve and make up to volume with diluent. Transfer 5mL of this solution into a 100mL volumetric flask and make up to the volume with diluent.

Exposure to white fluorescent light:

Transfer about 3000.0 mg of the Tolterodine sample into a petri dish, spread uniformly and expose to the white fluorescent light for 1.2 million LUX hours for a period of 23 hours.

After Exposure to white fluorescent light:

Weigh and transfer about 100mg of sample into a 100mL volumetric flask dissolve and make up to with diluent. Transfer 5mL of this solution into a 100mL volumetric flask and make up to the volume with diluent.

c) Exposure to UV light at 365 nm:

Transfer about 3000.0 mg of the Tolterodine sample into a petri dish, spread uniformly and expose to the UV light at 365 nm for 200 W/m² for a period of 48 hours.

After Exposure to UV light at 365 nm:

Weigh and transfer about 100mg of sample into a 100mL volumetric flask dissolve and make up to with diluent. Transfer 5mL of this solution into a 100mL volumetric flask and make up to the volume with diluent.

d) Exposure to Heat at 105°C:

Transfer about 3000.00 mg of the sample in a petri dish, spread uniformly and expose to heat at 105°C for ten days.

i) After 24 hours:

After twenty-four hours weigh and transfer about 100mg of sample into a 100mL volumetric flaskdissolve and make up to the volume with diluent. Transfer 5mL of this solution into a 100mL

volumetric flask and make up to the volume with diluent.

ii) After 10 days:

After ten days weigh and transfer about 100mg of sample into a 100mL volumetric flask dissolve and make up to with diluent. Transfer 5mL of this solution into a 100mL volumetric flask and make up to the volume with diluent.

RESULTS AND DISCUSSIONS:

Acid Hydrolysis:

Exposure conditions	At room temperature	At 60˚C temperature
Acid Hydrolysis	From the 2D plot at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.	The solution was injected after being exposed to temperatures of 60 ˚C for 3, 6, 12, and 24 hours. The heating treatment shows no degradation of the product at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle

Base Hydrolysis:

Exposure conditions	At room temperature	At 60˚C temperature
Base Hydrolysis	From the 2D plot at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.	The solution was injected after being exposed to temperatures of 60 ˚C for 3, 6, 12, and 24 hours. The heating treatment shows no degradation of the product at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle

Oxidation:

Exposure conditions	At room temperature	At 60˚C temperature
Oxidation	From the 2D plot at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.	The solution was injected after being exposed to temperatures of 60 ˚C for 3, 6, 12, and 24 hours. The heating treatment shows no degradation of the product at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

UV light degradation:

The solution was injected after being exposed to UV light at 365nm for 3, 6, 12, and 24 hours. From the 2D plot at 210nm no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine tartrate peak, this because the Purity threshold is greater than the purity angle. The representative purity and 3D chromatogram was showed in Figure.3.

Figure 3. Representative purity peak and 3D chromatogram of UV light degradation

Figure 4. Representative purity peak and 3D chromatogram of Heat degradation

Heat degradation:

The solution was injected after being exposed to temperatures of 60 ˚C for 3, 6, 12, and 24 hours. From the 2D plot at 210nm no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine tartrate peak, this because the Purity threshold is greater than the purity angle. The representative purity and 3D chromatogram was showed in Figure.4.

Solid state stability:

After Exposure to white fluorescent light:

The control sample and the sample prepared after exposure the sample to fluorescent light for a period of 23 hours was injected to the HPLC system contains PDA detector. No degradation of the product at 210nm indicates no additional peak was observed on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

After Exposure to UV light at 365 nm:

The control sample and the sample prepared after exposure the sample to UV light for a period of 48 hours was injected to the HPLC system contains PDA detector. No degradation of the product at 210 nm, shows no additional peak was observed on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

Exposure to Heat at 105°C:

After 24 hours:

The control sample and the sample prepared after exposure to 105°C for a period of 24 hours was injected to the HPLC system contains PDA detector. No degradation of the product at 210 nm, shows no additional peak was observed on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

After 10 days:

The control sample and the sample prepared after exposure to 105°C for a period of 10 days was injected to the HPLC system contains PDA detector. No degradation of the product at 210 nm, shows no additional peak was observed on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

CONCLUSION:

From the results obtained in this study, one can conclude that the Tolterodine is stable to all the conditions tested in this study, hence it can be conclude that the product shows no appreciable degradation in the conditions tested in this study.

Results also show that the HPLC method used in this study is able to detect any degradation of the product and separate any degradation impurities formed, thus making it a suitable stability indicating method.

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Benerjee patrudu, Gitam University, Hyderabad for providing the gift sample of Tolterodine and providing necessary facilities to carry out the research work with keen interest and help.

REFERENCES:

1. S Vijaya Bhaskar, J.V.L.N Seshagiri Rao, and Vijaya Sree. Development and Validation of an RP-HPLC method for the estimation of Tolterodine in Raw materials and Tablet dosage forms. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 2013; 4(4): 1378-1384.

2. C. Bala Kumar, B. Lakshmi Narayanan, M. Chandrasekar, P. Malairajan, E. P. Kumar. Development and Validation of RP-HPLC Method for the Quantitative Estimation of Tolterodine Tartrate in Capsule Formulation. RGUHS J Pharm Sci, 2013; 3: 58-64.

3. N. Ramathilagam, M. meeradevi, P. Solairaj, S.C. Rajesh. Development and validationof of stability indicating HPLC method for determination of tolterodine tartrate. International Journal of Pharmacy and Biological Sciences, 2012; 3: 332-337.

4. B. Siddartha, Dr. I. Sudheer Babu, A. Krupalini, V. Prathyusha. Development and Validation of UV– Spectrophotometric Method of Tolterodine in bulk and Pharmaceutical Dosage Form. Asian J. Pharm. Ana., 2013; 3: 102-104.

5. S. Ashutosh kumar, Manidipa debnath, J.V.L.N. Seshagiri rao. Stability Indicating RP-HPLC Method for the Determination of Tolterodine Tartrate in Bulk as well as in Pharmaceutical formulation. Int J Pharm Pharm Sci, 2013; 5: 665-671.

6. SK. Rihana Parveen, P. Srinivasa Babu, K.B. Chandrasekar, B.R. Challa. Analytical method development and validation of Tolterodine in pharmaceutical dosage forms by RP-HPLC. Der Pharmacia Lettre, 2014; 6:246-254.

7. A. S. Reddy, R. C. Reddy and P. Venkateswarlu, Journal of Chemical and Pharmaceutical Research. Identification, isolation and quantification of unknown impurity in tolterodine tartrate tablets by stability indicating HPLC method. 2012; 4: 3659-3664.

8. Sathish Kumar Shetty, Arpan Shah, Development and Validation of Tolterodine by RP-HPLC Method in Bulk Drug and Pharmaceutical Dosage Forms International Journal of Pharma Tech Research, 2011; 3:1083-1087.

Exposure conditions

At room temperature

At 60˚C temperature

Exposure conditions

At room temperature

At 60˚C temperature

From the 2D plot at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

Exposure conditions

At room temperature

At 60˚C temperature

From the 2D plot at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.

Exposure conditions

At room temperature

At 60˚C temperature

From the 2D plot at 210nm, no impurities can be noted on the chromatogram. Reviewing the PDA plots namely the purity plot, it can be said that there are no impurities that fall under the Tolterodine peak, this because the Purity threshold is greater than the purity angle.