INTRODUCTION:

Stability testing is used as a primary tool to determine and asses the expiration dating and storage conditions for Pharmaceutical products. Stability testing includes long-term and accelerated conditions, where the product is stored

at room temperature and humidity, high heat and controlled humidity conditions. Stability study results are the evidence of establishment and assurance of safety, quality and efficacy of the drug product. In order to determine the product stability, the appropriate physical, chemical, biological and microbiological testing must be performed.

One of the evaluation criteria is the appearance of impurities during real time and accelerated stability studies. Tolterodine Tartrate is described chemically as 2-(3-(diisopropylamino)-1- phenylpropyl)-4-methylphenol Tartrate (Fig.1). The Drug is listed in the Merck index. Tolterodine Tartrate is used for the treatment of urinary urge incontinence and other symptoms of overactive bladder [1]. 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 [2-5]^.A liquid chromatography–tandem mass spectrometry for Quantization of Tolterodine and its 5-hydroxy methyl metabolite in plasma samples was reported in the literature. Determination of Assay of Tolterodine Tartrate in Plasma, serum and urine by Capillary Gas chromatography coupled with Mass spectrometer was also reported in the literature. An enantio specific HPLC method for the determination of (S)-enantiomer impurities in (R)-Tolterodine Tartrate and Simultaneous Estimation of Tamsulosin HCL and Tolterodine Tartrate in pharmaceutical dosage form was also reported [6]. A HPLC method for determination of Tolterodine Tartrate in dosage forms was also available in literature for routine quality control samples [7]. A Validated Stability-Indicating HPLC Method for the determination of Related Components in Tolterodine Tartrate was also reported in the Literature [8]. The chemical structure of the Drug was represented in Fig. no.1

Fig.no.1 It shows the Chemical Structure of Tolterodine

MATERIAL AND METHOD:[9]

Chemicals and Reagents Used: The following chemicals were procured for the process Water [HPLC Grade], Tolterodine [Working Standard], Acetonitrile [HPLC Grade], Ortho phosphoric acid all the chemicals were procured from Standard Solutions, HCL procured from Finar Chemical Limited, NaOH procured from S D FINE- Chem Limited and H₂O₂ procured from ALPHA PHARMA Limited and Tolterodine Tablet 2mg was collected from the Local market, manufacturer Ranbaxy, brand name ROLITEN.

Apparatus and Chromatographic Parameters:

Equipment:High performance liquid chromatography equipped with Auto Sampler and DAD or UV detector.

Column: Symmetry C18 (4.6 x 150mm, 5 mm, Make: Kromosil)

Flow rate: 0.8mL per min

Wavelength: 282 nm

Injection volume: 20 ml

Column oven: Ambient

Run time: 5.0 min

Detector: Photo diode array

Soft ware: Empower 2

MFD by: Waters

Preparation of Phosphate buffer [10-11]:

The Buffer Solution was prepared by weighed accurately 7.0 grams of Potassium dihydrogen phosphate into a 1000ml beaker, dissolved and diluted to 1000ml with Water HPLC grade. The pH was adjusted to 3.0 by using Orthophosphoric acid.

Preparation of mobile phase:

The Mobile Phase was prepared with a ratio of 50:50 of Buffer and Acetonitrile. The Buffer was taken 500mL and Acetonitrile was taken 500 mL and degassed in ultrasonic water bath for 5 minutes. Then it was filtered through 0.45 µ filter under vacuum filtration.

Diluent Preparation:

The Mobile phase was used as diluent.

Preparation of the Tolterodine Standard and Sample Solution:

Standard Solution Preparation:

The Standard Stock Solution was prepared by weighed accurately and transferred 10mg of Tolterodine [Working standard] into a 10 mL volumetric flask added, about 7 mL of Diluent and sonicated to dissolved it completely and the volume was made up to the mark with the same solvent. Further from the above prepared Stock Solution pipette out 0.6 ml into a 10ml volumetric flask and diluted up to the mark with diluent. The resultant solution was mixed well and then it was filtered through 0.45µm filter.

Sample Solution Preparation:

The Sample Stock Solution was prepared by weighed accurately 5 Tolterodine Tablets and calculated the average weight. The drug sample was accurately weighed equivalent to 10 mg and transferred into a 10 mL volumetric flask. About 7 mL of diluent was added and sonicated to dissolve it completely and then the volume was made up to the mark with the diluent. The resultant solution was mixed well and it was filtered through 0.45µm filter. Further from the above prepared Stock Solution pipette out 0.6 ml into a 10ml volumetric flask and diluted up to the mark with diluent. The resultant solution was mixed well and then it was filtered through 0.45µm filter.

Standard and Sample Solutions Injected to the Column [Chromatographic System]:

About 20mL of standard and sample solutions were injecting to the chromatographic system and the area for the drug was measured and the % Assay was calculated by using suitable formulae.

System Suitability [12-14]:

The Tailing factor for the peak due to Tolterodine in Standard solution should not be more than 2.0. The Theoretical plates for the Tolterodine peak in Standard solution should not less than 2000.

Formula for calculating the Assay for the Drug Tolterodine:

AT WS DT P Avg. Wt.

Assay % =--------X--------X-------X--------X-----------------X 100

AS DS WT 100 Label Claim

Where:

AT = Peak Area of Tolterodine obtained with test preparation

AS = Peak Area of Tolterodine obtained with standard preparation

WS = Weight of working standard taken in mg

WT = Weight of sample taken in mg

DS = Dilution of Standard solution

DT = Dilution of sample solution

P = Percentage purity of working standard

Results obtained for the System Suitability:

1) The Tailing factor obtained from the standard injection was found to be1.6

2) The Theoretical Plates obtained from the standard injection was found to be 2067.6

Assay Results for Tolterodine:

634803 10 0.4 10 10 99.8 232.4

Assay % =---------X--------X------X--------X-----X-------X------X 100

634803 10 10 233.1 0.4 100 8

= 100.2%

VALIDATION DEVELOPMENT [15-18]:

1. Precision:

The precision of an analytical procedure expresses the closeness of measurements obtained from multiple sampling of the same homogenous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision and reproducibility. The precision of an analytical procedure is usually expressed as the variance, standard deviation or coefficient of variation of a series of measurements. The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. The data was represented in Table no.1

Table no.1The results for Precision were summarized for the drug Tolterodine.

Injection	Area
Injection-1	667103
Injection-2	667059
Injection-3	669293
Injection-4	670508
Injection-5	671724
Average	669137
Standard Deviation	2064.7
%RSD	0.31

Acceptance Criteria:

The % RSD for the area of five standard injections results should not be more than 2%.

2. Intermediate Precision (Ruggedness):

To evaluate the intermediate precision, precision was performed on different day by using different make column of same dimensions. The standard solution was injected for five times and the area was measured for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits. The Data was represented in Table no. 2

Table no. 2The results were summarized for Intermediate

Precision (Ruggedness) for the drug Tolterodine.

Injection	Area
Injection-1	629965
Injection-2	630893
Injection-3	632215
Injection-4	633029
Injection-5	633771
Average	631975
Standard Deviation	1549.6
%RSD	0.25

Acceptance Criteria:

The % RSD for the area of five standard injections results should not be more than 2%.

3. Accuracy:

The accuracy of an analytical procedure expressed the closeness of agreement between the value which was accepted either as a conventional true value or an accepted reference value and the value found. The Standard and Sample were injected to the chromatographic system for Accuracy 50%, 100% and 150%. The amount found was calculated and the amounts added for Tolterodine were calculated. The individual recovery and mean recovery values were also calculated. The Data was represented in Table no.3.

Acceptance Criteria:

The % Recovery for each level should be between 98.0 to 102.0%.

4. Linearity:

The linearity of the analytical procedure was its ability to obtain the test results which are directly proportional to the concentration (amount) of analyte in the sample. Different levels were prepared and all Levels were injected individually into the chromatographic system and the peak area was measured. A graph was plotted for peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and the correlation coefficient was calculated. The Data were represented in Table no. 4.

Table no.3 The Accuracy results were summarized for the drug Tolterodine.

%Concentration (at specification Level)	Area	Amount Added (mg)	Amount Found (mg)	% Recovery	Mean Recovery
50%	319003	5.0	5.0	100.2%	99.0%
100%	628847	10.0	9.87	98.7%
150%	936919	15.0	14.7	98.1%

Table no. 4The Linearity Results were summarized for the drug Tolterodine:

Sl. No.	Linearity Level	Concentration	Area
1	I	20µg/ml	236168
2	II	40µg/ml	429629
3	III	60µg/ml	675098
4	IV	80µg/ml	827109
5	V	100µg/ml	1068003
Correlation Coefficient			0.999

Acceptance Criteria:

The Correlation coefficient should be not less than 0.999.

5. Limit of Detection:

The detection limit of an individual analytical procedure was the lowest amount of analyte in a sample which can be detected but not necessarily quantities as an exact value.

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank : 42µV

Signal Obtained from LOD solution (1.8% of target assay concentration) : 122 µV

S/N =1223/42= 2.90

Acceptance Criteria:

The S/N Ratio value should be 3 for LOD solution.

6. Limit of Quantification:

The Quantification limit of an individual analytical procedure was the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy.

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank 42µV

Signal Obtained from LOQ solution (6.0% of target assay concentration) 418 µV

S/N = 418/42 = 9.95

Acceptance Criteria:

The S/N Ratio value should be 10 for LOQ solution.

7. Robustness:

The robustness of an analytical procedure was a measured of its capacity to remain unaffected by small, but deliberate variations in method parameter and provides an indication of its reliability during normal usage.

a) The flow rate was varied at 0.6 to1.0 ml/min.:

The Standard solution 60 µg/ml was prepared and analysed using the varied flow rates along with actual flow rate.On evaluation of the above results, it was concluded that the variation in flow rate does not affected the method significantly. Hence it indicates that the method was robust even by change in the flow rate ±10%. The Data was represented in Table no. 5

Table no. 5The results were summarized for the System Suitability with change in the flow rate for the drug Tolterodine.

Sl. No.	Flow Rate (ml/min)	System Suitability Results
Sl. No.	Flow Rate (ml/min)	USP Plate Count	USP Tailing
1	0.6	2086.9	1.7
2	0.8	2067.6	1.6
3	1.0	2020.5	1.5

b) The Organic composition in the Mobile phase was varied from45% to 55%:

The Standard solution 60 µg/ml was prepared and analysed using the varied Mobile phase composition along with the actual mobile phase composition in the method. On evaluation of the above results, it was concluded that the variation in 10% Organic composition in the mobile phase does not affected the method significantly. Hence it indicates that the method is robust even by change in the Mobile phase ±10%. The Data was represented in Table no. 6.

Table no.6The results were summarized for System Suitability with change in Organic Composition for the drug Tolterodine.

S No.	Change in Organic Composition in the Mobile Phase	System Suitability Results
S No.		USP Plate Count	USP Tailing
1	10% less	2443.9	1.6
2	Actual	2067.6	1.6
3	10% more	2166.3	1.7

8. Degradation studies [19-20]:

The International Conference on Harmonization (ICH) guideline entitled stability testing of new drug substances and products requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance. The aim of this work was to perform the stress degradation studies on the Tolterodine using the proposed method.

a. Hydrolytic degradation under acidic condition:

0.6 ml of stock solution of Tolterodine was prepared and taken in a 10ml volumetric flask further 3 ml of 0.1N HCl was added. Then the volumetric flask was kept at normal condition for 90 minutes and then it was neutralized with 0.1 N NaOH and the volume was made upto the mark with the diluent [10ml]. Then the resultant solution was filtered with 0.65 microns syringe filters and placed in the vials.

b. Hydrolytic degradation under alkaline condition:

0.6 ml of stock solution of Tolterodine was prepared and taken in a 10ml volumetric flask further 3 ml of 0.1N NaOH was added. Then the volumetric flask was kept at normal condition for 90 minutes and then it was neutralized with 0.1 N HCL and the volume was made up to mark [10ml] with the diluent. Then the resultant solution was filtered with 0.65 microns syringe filters and placed in the vials.

c. Thermal induced degradation:

0.6 ml of stock solution of Tolterodine was prepared and taken in a 10ml volumetric flask further 3 ml of the diluent was added. Then the volumetric flask was kept at reflex condition for 60 minutes and the volume was made upto the mark [10ml] with the diluent. Then the resultant solution was filtered with 0.65 microns syringe filters and placed in the vials.

d. Oxidative degradation:

0.6 ml of the stock solution of Tolterodine was prepared and taken in a 10ml volumetric flask further 1 ml of 3 % w/v of hydrogen peroxide was added. The volume was made up to the mark [10ml] with diluent. The volumetric flask was then kept at room temperature for 15 min. The resultant solution was filtered with 0.65 microns syringe filters and placed in the vials.

e. Photolytic degradation:

The sample of Tolterodine was exposed to near ultra violet lamp in photostablity chamber providing illumination for 1hr, 5hr. From the above sample 10 mg of the sample was dissolved in water and volume made up to mark [10 ml]. From the above solution dilutions were made to achieve the appropriate concentration (30μg/ml) and the solution was taken in the vials. The following results were obtained for the drug Tolterodine under Force degradation studies which were summarized in Table no. 7.

RESULT AND DISCUSSION:

The present study was carried out to develop a sensitive, precise and accurate stability indicating RP-HPLC method for the analysis the drug in Bulk as well as in pharmaceutical formulations or dosage forms. In order to method development under isocratic conditions, a mixtures of Phosphate Buffer [whose pH was adjusted to 3 with Orthophosphoric acid] and Acetonitrile [HPLC grade] were taken in different combinations and tested as mobile phase on a Symmetry C18 (4.6 x 150mm, 5 mm, Make: Kromosil) column. A binary mixture of Phosphate Buffer with pH 3 and Acetonitrile HPLC grade in 50:50 v/v proportion was proved to be the most suitable of all combinations since the chromatographic peaks were better defined and resolved and almost free from tailing. The retention time obtained for the drug Tolterodine was around 2.896 min. A model chromatogram was shown in Fig. no.2.

Fig.no.2 A model chromatograph for the Drug Tolterodine.

The Precision data was represented in Table no. 1. The accuracy of an analytical procedure expresses the closeness of agreement between the value which was accepted either as a conventional true value or an accepted reference value and value found. The data found was in specified limits and it was represented in Table no.3. In order to test the linearity of the method, five dilutions of the working standard solutions of the drug in the range of 20 to 100µg per mL were prepared. The data was represented in Table no. 4. Each of the dilutions was injected into the column [Chromatographic system] and the graph for the Linearity Curve was represented in Fig no. 3.

Fig.no.3 Shows Linearity Curve for the Drug Tolterodine.

The method was duly validated by evaluation of the required parameters. When Tolterodine was analyzed by the proposed method in the intra and inter-day (Ruggedness) variation results, a low coefficient of variation was observed and it was represented in Table no. 2. It shows that the present HPLC method was highly precised and it was represented by Fig no. 4.

Fig.no.4 It shows the Ruggedness of the Chromatograph for the Drug Tolterodine.

Robustness of the method was found out by testing the effect of small deliberate changes in the chromatographic conditions and the corresponding peak area. The factors selected for this purpose were flow rate and percentage composition variation in the mobile phase. The method was found to be robust enough that the peak area was not apparently affected by small variation in the chromatographic conditions. The Fig. no. 5, 6, 7 and 8 were represented the System Suitability.

Fig.no.5 It shows the System Suitability results with decrease in the flow rate for the Drug Tolterodine.

Fig.no.6 It shows the System Suitability with increase in the flow rate for the Drug Tolterodine.

Fig.no.7 It shows the System Suitability with decrease in the composition of the mobile phase for the Drug Tolterodine.

Fig.no. 8 It shows the System Suitability with increase in the composition of the mobile phase for the Drug Tolterodine.

The system suitability parameters were within the limits as shown in Table 5 and 6. The Limit of detection and limit of quantification of the method were calculated based on standard deviation of the response and the slope (s) of the calibration curve at approximate levels of the limit of detection and limit of quantification. The LOD for the drug Tolterodine was found to be 0.108µg/ml and LOQ for the drug Tolterodine was found to be 0.36μg/mL.

In order to evaluate the stability of Tolterodine and ability of the method to separate Tolterodine from its degradation products, the drug Tolterodine was subjected to various stress conditions such as Hydrolytic degradation under acidic condition (using 0.1N HCl and 0.1 N NaOH), Hydrolytic degradation under alkaline condition (using0.1N NaOH and 0.1N HCL), Thermal induced degradation (Reflex Condition for 60 mins), Oxidative degradation (by using 3 % w/v of hydrogen peroxide), Photolytic degradation (exposed to near ultra violet lamp in photostablity chamber providing illumination for 1hr, 5hr). The following chromatograph represents the degradation studies for the drug [Tolterodine] which were represented in fig. no.9, 10, 11, 12 and 13.

Fig. no.9. The chromatograph represents the Hydrolytic degradation under acidic condition.

Fig. no.10. The chromatograph represents the Hydrolytic degradation under alkaline condition.

Fig. no. 11.The chromatograph represents the Thermal induced degradation.

Fig. no. 12.The chromatograph represents the Oxidative degradation.

Fig. no. 13. The chromatograph represents the Photolytic degradation.

The drug content formulations were quantified by using the proposed analytical method. The low coefficient of variation in the recovery data was indicated that the reproducibility of the method in dosage forms. It was concluded that the proposed HPLC method is sufficiently sensitive and reproducible for the analysis of Tolterodine in the Tablet formulation dosage form within a short analysis time.

CONCLUSION:

It was concluded that the proposed stability indicating RP-HPLC method developed for the quantitative determination of Tolterodine in bulk as well as in its formulations was simple, selective, sensitive, accurate, precise and rapid. The method was proved to be superior to most of the reported methods. The mobile phases were simple to prepare and economical. The sample recoveries in the formulation were in good agreement with their respective label claims and they suggested non-interference of formulation excipients in the estimation. Hence the method can be easily adopted as an alternative method to report routine determination of Tolterodine depending upon the availability of chemicals and nature of other ingredients present in the sample. The method also finds use in clinical, biological and pharmacokinetic studies for the drug Tolterodine. The method was validated as per ICH guidelines, and validation acceptance criteria were met in all cases. Application of this method for estimation of Tolterodine from tablet dosage form and stressed samples showed that neither the degradation products nor the excipients interfered in the estimation of drug. Hence, this method was specific, stability-indicating and can be successfully used for the estimation of Tolterodine in bulk and pharmaceutical dosage forms.

REFERENCE:

1 http://en.wikipedia.org/wiki/Tolterodine

2. http://www.rxlist.com/detrol-la-drug.htm

3. National center of Biotechnology information. Available at: http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001025/ (access on April 2012).

4. ICH Harmonized Tripartite Guideline on validation of Analytical Procedures: Q 2 (R1) (2005).

5. United States Pharmacopeia Asian edition (2005).

6. Supriya M Mhamunkar, Roshani Y. Vyavaharkar and Suvarna I Bhoir; RP-HPLC Method Development and Validation for the Simultaneous Estimation of Tamsulosin HCL and Tolterodine Tartrate in pharmaceutical dosage form; Int J Pharm Pharm Sci. 2012; 4 Suppl 5:320-322.

7 Dwibhashyam V.S, Keerthi P, Ratna J. V, Nagappa A. N; Reverse-Phase High performance liquid chromatographic method for the determination of Tolterodine Tartrate in routine quality control samples. PDA J Pharm Sci Technol.2009; 63(3):234-239.

8. S Radha Krishna, B.M. Rao and N. Someswara Rao, A Validated Stability-Indicating HPLC Method for the Determination of Related Substances and Assay of Tolterodine Tartrate. Rasayan J. Chem.2009; 2(1):144-150.

9 S. Ashutosh Kumar, Manidipa Debnath, J.V.L.N.Seshagiri Rao, Method Development and Validation of Tolterodine Tartrate in Bulk as well as in Pharmaceutical Formulation by Using RP-HPLC; Int J Pharm Pharm Sci, Vol 5, Issue 3, 665-671.

10 Sethi, P.D., In; HPLC ‘High Performance Liquid Chromatography’, Quantitative Analysis of Pharmaceutical Formulations, 1st Edn; CBS Publishers and Distributors, New Delhi, 2001, 3-72,116-120.

11 Indian Pharmacopeia 2007, Volume I: The Indian Pharmacopoeia Commission, 477-478.

12 Yuri Kazakevich and Rosario Lobrutto Seton: HPLC for Pharmaceutical Scientists 1^st ed, Wiley-VCH; 2007; 369-382.

13 Stavros Kromidas. HPLC Made to Measure, A Practical Book for Optimization. Published by Wiley-VCH; 2006; 62-66.

14 Yuri Kazakevich and Rosario Lobrutto Seton: HPLC for Pharmaceutical Scientists.1st ed, Wiley-VCH; 2007; 389-391.

15 International Conference on Harmonization (ICH). Topic Q2 (R1). Validation of analytical procedures: Text and Methodology. Geneva, Switzerland November 2005.

16 Sharma B. K.; Instrumental Method of Chemical Analysis, 21th Edition, Goel Publishing Housing, Krishna Prakashan Ltd., 2002; 3.

17 Skoog, West and Holler; Fundamental of Analytical Chemistry, 7th Edition, Saunders college Publishing, 1992.

18 Conners K. A.; Textbook of Pharmaceutical Analysis, 3 rd Edition, A Wiley- Intersciences Publication, 1999; 616-622.

19 ICH harmonized tripartite guideline. Impurities in New Drug products Q3B (R2) current step 4 version dated 2 June 2006.

20 ICH, Q1A (R2), Stability Testing of New Drug Substances and Products, Geneva, Feb.2003.

Received on 19.06.2013 Modified on 05.07.2013

Asian J. Research Chem. 6(7): July 2013; Page 694-701

Sl. No.	Degradation Studies	Retention Time	Area	Height	USP Plate Count	USP Tailing Factor	Purity Angle	Purity Threshold
1	Hydrolytic degradation under acidic condition	2.916	591464	62646	2146.8	1.6	0.29	0.38
2	Hydrolytic degradation under alkaline condition	2.915	578744	61298	2241.5	1.6	0.31	0.42
3	Thermal induced degradation	2.916	559665	59277	2328.6	1.6	0.36	0.38
4	Oxidative degradation	2.916	534226	56583	2465.8	1.6	0.21	0.26
5	Photolytic degradation	2.918	558624	59206	2153.6	1.6	0.15	0.21